PAS kinase

ABSTRACT

The invention provides methods and compositions relating to a novel kinase designated PAS Kinase (PASK). The compositions include isolated polypeptides comprising a native PASK protein or a PASK N-terminal domain and polypeptides consisting of a PASK PAS-A or PAS-B domain, as well as isolated polynucleotides encoding such polypeptides, and expression vectors and cells comprising such polynucleotides. The methods include binding assays comprising the steps of incubating a mixture comprising a subject polypeptide with a ligand under conditions wherein the polypeptide binds the ligand; and detecting binding of the polypeptide to the ligand.

FIELD OF THE INVENTION

The field of this invention is protein binding assays.

BACKGROUND OF THE INVENTION

We have identified a novel enzyme designated PAS-kinase (PASK). The human version of PASK is encoded by a single gene located on chromosome 2, band q37. Analysis of genomic and cDNA clones of PASK has shown the gene to be composed of 18 coding exons covering roughly 37 kilobases of human chromosome 2. The enzyme is 1,323 amino acid residues in length and highly related in primary sequence to polypeptides specified in the genomes of flies and yeast. RT-PCR assays conducted on multiple tissues of the mouse indicate that PASK mRNA is expressed at a similar level in all tissues.

As shown in Table 1 (below), the polypeptide sequences of the human, fly and yeast versions of PASK are most conserved in three regions. Two of the three conserved regions correspond to PAS domains, designated PAS-A and PAS-B, the former being located closer to the amino terminus of the enzyme. The third conserved region of PASK encodes a serine/threonine kinase domain. When compared with all entries available in the database of protein kinases kept by Tony Hunter [1], PASK is most similar in primary amino acid sequence to the catalytic domains of the 5′ AMP activated protein kinase (AMPK), its yeast ortholog SNF1, and the product of the Pim-1 oncogene.

We conducted a two-pronged approach to resolve the biological function of PASK. Biochemical experiments were carried out to study the catalytic activity of the enzyme as well as the modulatory role enacted by the two PAS domains. Complementary genetic experiments were undertaken to study the biological role of PASK in budding yeast. The latter experiments have also directed preliminary studies pertinent to the role of PASK in mammalian cells. In summary, these efforts have established PASK as a serine/threonine kinase that is regulated in cis by its two PAS domains. They have likewise implicated PASK in the regulation of translation and the balance of cell growth (cell size) and mitosis. Biophysical studies of the two PAS domains of PASK have identified PAS-A as a ligand-binding regulatory domain of the PASK enzyme. Together these studies reveal an entirely novel and unanticipated regulatory system that represents a valuable target for the development of synthetic organic compounds capable of regulating the mitotic growth of mammalian cells.

Limited aspects of this disclosure were presented at the 19th International Conference on Magnetic Resonance in Biological Systems at Florence, Italy in August 2000; see, Structural studies of PAS domains: Insight into the link between ligand and protein binding, K H Gardner, C A Amezcua and S M Harper, Aug. 25, 2000, 19^(th) Intrnl Conf Magnetic Resonance in Biological Systems, Florence, Italy; and Solution structure and dynamics of a eukaryotic PAS domain: Evidence for a flexible ligand binding region, C A Amezcua, S M Harper and K H Gardner, Aug. 21, 2000, 19^(th) Intrnl Conf Magnetic Resonance in Biological Systems, Florence, Italy. Genbank accession KIAA0135 has sequence similarity to the disclosed human PASK.

SUMMARY OF THE INVENTION

The invention provides methods and compositions relating to a novel kinase designated PAS Kinase (PASK). The compositions include isolated polypeptides comprising a PASK N-terminal domain, particularly a native PASK protein, and polypeptides consisting of, or consisting essentially of, a PASK PAS-A or PAS-B domain, as well as isolated polynucleotides encoding such polypeptides, and expression vectors and cells comprising such polynucleotides.

The subject methods include binding assays comprising the steps of incubating a mixture comprising a subject polypeptide with a ligand under conditions wherein the polypeptide binds the ligand; and detecting the binding of the polypeptide to the ligand. In particular embodiments, the assay is a kinase assay wherein the ligand is a substrate, the mixture further comprises a nucleoside triphosphate, the binding effects phosphorylation of the substrate, and the detecting step comprises detecting the phosphorylated substrate. In other embodiments, the assay is a NMR-based assay wherein the detecting step comprises detecting an NMR shift in the mixture.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows an alignment of the PASK PAS-A domains of human (amino acids 131-237 of SEQ ID NO:2), fly (amino acids 72-177 of SEQ ID NO:4) and yeast (amino acids 460-564 of SEQ ID NO:6 and amino acids 288-397 of SEQ ID NO:8).

FIG. 1B shows an alignment of the PASK PAS-B domains of human (amino acids 341-439 of SEQ ID NO:2), fly (amino acids 280-375 of SEQ ID NO:4) and yeast (amino acids 744-843 of SEQ ID NO:6 and amino acids 522-621 of SEQ ID NO:8).

FIG. 1C shows an alignment of the PASK kinase domains of human (amino acids 1005-1251 of SEQ ID NO:2), fly (amino acids 582-828 of SEQ ID NO:4) and yeast (amino acids 1102-1354 of SEQ ID NO:6 and amino acids 847-1099 of SEQ ID NO:8). The three phosphorylatable activation loop residues are marked with a star.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The nucleotide sequences of cDNAs encoding native PASK polypeptides from human, D. Melanogaster, and yeast (two variants) are shown as SEQ ID NOS:1, 3, 5 and 7, respectively, and the full translates are shown as SEQ ID NOS:2, 4, 6 and 8, respectively. As described in further detail below, these translates comprise an N-terminal functionality, PAS-A and PAS-B domains and a kinase domain.

The subject polypeptides are isolated or pure: an “isolated” polypeptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5%, more preferably at least about 50% by weight of the total polypeptide in a given sample and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. The polypeptides and polypeptide domains may be synthesized, produced by recombinant technology, or purified from mammalian, preferably human cells. They may be joined, covalently or noncovalently, with a wide variety of conjugates, including labels, tags, etc., particularly other polypeptide sequences. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art.

PASK N-terminal domain polypeptides, including the human PASK N-terminal domain (residues 1-89 of SEQ ID NO:2)and functional fragments thereof, elicit PASK specific antibody in a heterologous host (e.g. a rodent or rabbit), etc. Accordingly, these polypeptides provide PASK-specific antigens and/or immunogens, especially when coupled to carrier proteins (see, e.g. Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory). For example, polypepetides corresponding to PASK-specific N-terminal domain subsequences are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of PASK-specific antibodies is assayed by solid phase immunosorbant assays using immobilezed PASK polypepetides of SEQ ID NO:2, see, e.g. Table 1.

Table 1. Immunogenic N-terminal PASK polypepetides eliciting PASK-specific rabbit polyclonal antibody: PASK polypeptide-KLH conjugates immunized per protocol described above.

PASK Polypeptide Sequence Immunogenicity SEQ ID NO: 2, residues 1-10 +++ SEQ ID NO: 2, residues 12-21 +++ SEQ ID NO: 2, residues 20-29 +++ SEQ ID NO: 2, residues 32-49 +++ SEQ ID NO: 2, residues 52-61 +++ SEQ ID NO: 2, residues 65-79 +++ SEQ ID NO: 2, residues 77-89 +++

The amino acid sequences of the subject polypeptides are used to back-translate polypeptide-encoding nucleic acids optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate oligonucleotide primers and probes for use in the isolation of natural PASK polypeptide-encoding nucleic acid sequences (“GCG” software, Genetics Computer Group, Inc, Madison Wis.). PASK polypeptide-encoding nucleic acids are used in expression vectors and incorporated into recombinant host cells, e.g. for expression and screening, transgenic animals, e.g. for functional studies such as the efficacy of candidate drugs for disease associated with PASK-modulated cell function, etc.

The invention also provides nucleic acid hybridization probes and replication/amplification primers having a PASK N-terminal domain encoding cDNA sequence or fragments thereof sufficient to effect specific hybridization thereto (i.e. specifically hybridize with the corresponding PASK N-terminal domain cDNA). Such primers or probes are at least 12, preferably at least 24, more preferably at least 36 and most preferably at least 96 bases in length. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5×SSPE (0.18 M NaCl, 0.01 M NaPO₄, pH7.7, 0.001 M EDTA) buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5×SSPE buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSPE buffer at 42° C. PASK nucleic acids can also be distinguished using alignment algorithms, such as BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410).

Table 2. Exemplary PASK nucleic acids which hybridize with a strand of SEQ ID NO:1 under Conditions I and/or II.

PASK Nucleic Acids Hybridization SEQ ID NO: 1, nucleotides 1-24 + SEQ ID NO: 1, nucleotides 25-48 + SEQ ID NO: 1, nucleotides 49-72 + SEQ ID NO: 1, nucleotides 73-96 + SEQ ID NO: 1, nucleotides 97-120 + SEQ ID NO: 1, nucleotides 121-144 + SEQ ID NO: 1, nucleotides 145-168 + SEQ ID NO: 1, nucleotides 169-192 + SEQ ID NO: 1, nucleotides 193-216 + SEQ ID NO: 1, nucleotides 217-240 + SEQ ID NO: 1, nucleotides 242-265 + SEQ ID NO: 1, nucleotides 391-711 + SEQ ID NO: 1, nucleotides 1021-1317 +

The subject nucleic acids are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Recombinant nucleic acids comprising native PASK cDNAs, or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by (i.e. contiguous with) a non-native sequence (a sequence other than that which it is joined to on a natural chromosome), or immediately flanked by a native flanking region of fewer than 2000, preferably fewer than 500, more preferably fewer than 100 nucleotides. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.

The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of PASK genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional PASK homologs and structural analogs. In diagnosis, PASK hybridization probes find use in identifying wild-type and mutant PASK alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic PASK nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active PASK.

The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of a PASK modulatable cellular function. Generally, these screening methods involve assaying for compounds which modulate the interaction of a subject PASK polypeptide with a ligand and/or natural binding target. A wide variety of assays for binding agents are provided including labeled in vitro protein-protein binding assays, immunoassays, cell based assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.

In vitro binding assays employ a mixture of components including a PASK polypeptide, which may be part of a fusion product with another peptide or polypeptide, e.g. a tag for detection or anchoring, etc. The assay mixtures comprise a ligand, which term is used generically to encompass specific binding targets including substrates, preferably small molecule ligands as opposed to large molecule ligands such as protein ligands. In a particular embodiment, the binding target is a substrate of PASK kinase activity. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like ATP or ATP analogs (for kinase assays), salts, buffers, neutral proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the PASK polypeptide specifically binds the ligand with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening.

After incubation, the agent-biased binding between the PASK polypeptide and one or more ligands is detected by any convenient way. For PASK kinase assays, ‘binding’ is generally detected by a change in the phosphorylation of a substrate. In this embodiment, kinase activity may be quantified by the transfer to the substrate of a labeled phosphate, where the label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, etc. A variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc.

A difference in the binding affinity of the PASK polypeptide to the ligand in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the polypeptide to the ligand. A difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.

The following experimental section and examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Biochemical properties of the PASK enzyme. A recombinant baculovirus was engineered to overexpress human PASK enzyme in cultured insect cells. The enzyme was expressed as a fusion with a His₆ tag at its amino terminus such that the recombinant polypeptide could be purified by Ni-affinity chromatography. Routine enzyme preparations were harvested from 0.8 liter of cultured Sf-9 cells. Cells were lysed by dounce homogenization in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.04% β-mercaptoethanol (“lysis buffer”) and spun at 100,000×G for one hour to remove insoluble debris. Soluble material (˜50 ml) was then applied to a 3 ml bed volume of Ni-affinity resin. Following application and 50× column volume wash, bound material was eluted with storage buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM DTT, 1 mM EDTA) supplemented with 250 mM imidazole. Eluted material was sequentially subjected to Mono-Q, anion-exchange chromatography, and eluted with increasing NaCl concentration. Fractions were evaluated by SDS-PAGE to identify PASK enriched fractions, pooled and dialyzed against storage buffer supplemented with 10% glycerol. Typical enzyme preparations yield 0.5 mg of intact PASK enzyme judged to be greater than 95% pure by visual inspection of Coomassie-stained SDS-PAGE gels.

Purified PASK was observed to become phosphorylated when exposed at room temperature to ATP and magnesium. Protein dilution studies revealed that the level of PASK phosphorylation diminished significantly upon dilution, indicating that phosphorylation occurs in trans. As will be shown subsequently, phosphorylation significantly elevates the activity of PASK by enhancing its K_(m) for substrate without altering the catalytic rate constant of the enzyme. Mass spectrometry was employed to identify the sites of auto-phosphorylation suffered by PASK in response to ATP. The major sites of phosphorylation are restricted to two short sequences, one corresponding to the putative activation loop of the, catalytic domain and another corresponding to a serine-rich segment of the polypeptide located on the immediate carboxyl terminal side of the catalytic domain. Roughly 2 moles of phosphate were observed to modify a tryptic peptide encompassing the putative activation loop of PASK, and an additional 3-4 moles of phosphate were observed to modify the C-terminal, serine-rich segment of the enzyme.

Kinetic data were used to characterize the activities of PASK enzyme samples derived without modification from baculovirus and following pre-phosphorylation in the presence of ATP. Enzyme activity was monitored using a synthetic substrate bearing the sequence NH₂-AMARAASAAALARRR-CO₂H (AMARA peptide, SEQ ID NO:9). The serine residue in this peptide is known to be phosphorylated by both AMPK and Snf1 [2], both of which are exceedingly similar in primary amino acid sequence to the catalytic domain of PASK. The three arginine residues located at the carboxyl terminus of the AMARA peptide allow avid binding to phosphocellulose paper, thereby facilitating quantitation of ³²P incorporation catalyzed by PASK from [γ-³²P]ATP. Whereas the observed V_(max) for the two enzyme samples was equivalent, K_(m) for substrate differed significantly. The prephosphorylated enzyme was observed to have a 6.5-fold lower K_(m) for substrate than the unmodified enzyme (Table 3). Moreover, lambda phosphatase treatment generated an enzyme which had 7-fold lower activity than the unmodified enzyme, and this treatment was fully reversible by allowing the enzyme to autophosphorylate in the presence of ATP.

Having mapped sites of phosphorylation to two segments of PASK, mutated derivatives of the enzyme were produced as a means of assessing the effects of phosphorylation on enzyme activity. The putative activation loop of PASK can be identified according to extensive studies on AMPK, one of the two closest relatives of PASK. It is known that threonine residue 172, located within the activation loop of AMPK, must be phosphorylated in order to support significant catalytic activity [3]. Mutated derivatives of PASK were generated wherein serine residue 1149, threonine residue 1161 (which aligns exactly with T172 of AMPK), and threonine residue 1165 were individually changed to alanine. All three of these residues map to a tryptic peptide that is autophosphorylated by PASK. As shown in Table 3, the S to A mutation at residue 1149 (S1149A) did not significantly alter either the catalytic rate or K_(m) for substrate. The T to A mutation at residue 1165 (T1165A) completely eliminated all detectable activity. Finally, the T to A mutation at residue 1161 (T1161A) led to the formation of an enzyme that was insufficiently stable to be purified from baculovirus and tested for enzymatic activity. Given the observation that two moles of phosphate were detected by mass spectrometric analysis of the tryptic peptide covering the activation loop of PASK, we tentatively conclude that the fully active enzyme is phosphorylated on threonine residues 1161 and 1165. These data indicate that PASK can, in trans, phosphorylate itself in a manner that mobilizes the activation loop of the enzyme and substantially enhances the avidity of the enzyme for substrate.

TABLE 3 Kinetic constants, K_(m) and V_(max), for wildtype and mutant forms of PASK using peptide substrate. Pre-phosphorylated Untreated Enzyme K_(m)(mM) SE V_(max) SE K_(m)(mM) SE V_(max) SE wildtype 0.2 0.05 0.2 NS 1.3 0.3 0.1 NS S1149A 0.6 0.05 0.1 NS T1161A N/A T1165A Catalytically inactive S1273, 77, 80A 0.3 NS 0.4 NS S1287, 89A 0.2 NS 0.1 NS S1273-1289A 0.2 NS 0.1 NS PASK ΔN 0.3 NS 2.8 NS

For this data table, enzyme assays were performed as described herein. PASK concentrations (0.5-2 μg/ml) were determined to have linear rates with respect to protein concentration and time. Kinetic constants were determined using peptide substrate concentrations between 0.06-1 mM. The data were fit to a non-linear Michaelis-Menten equation using the GraphPad Prism 2.0 program. V_(max) values given are μmol/min/mg. NS indicates an SE less than 0.05.

Three additional variants of the enzyme were engineered, expressed and studied in functional assays to assess the potential relevance of phosphorylation in the serine-rich, carboxyl-terminal domain of PASK. One mutant changed serine residues 1273, 1277 and 1280 to alanine (S1273,77,80), a second mutant changed serine residues 1287 and 1289 to alanine (S1287,89A), and a third mutant simultaneously changed all five serines to alanine (S 1273-89A). Each of the three mutated enzymes were expressed in baculovirus, purified and tested for both substrate avidity and catalytic rate. All three variants exhibited activities similar to the native enzyme.

A final series of experiments was conducted to investigate the influence of the amino terminal segment of PASK on catalytic activity of the enzyme. The only sequence conservation identifiable among the amino terminal segments of human, fly and yeast enzymes are two PAS domains (FIGS. 1A-1C). A truncated variant lacking both PAS domains (PASK ΔN) was engineered and expressed in baculovirus-infected Sf-9 cells. This variant of the human enzyme removed 948 residues on the amino-terminal side of the catalytic, serine/threonine kinase domain, and was expressed with a His₆ tag at the amino terminus of the truncated polypeptide. Although K_(m) for substrate was unaffected, this truncated variant exhibited a catalytic rate constant (V_(max)) 18-fold higher than the native enzyme (Table 3). In addition to this apparently derepressed basal activity, the truncated enzyme is not subject to regulation by phosphorylation. In contrast to the native enzyme, the lambda phosphatase-treated enzyme and the enzyme pre-incubated with magnesium and ATP showed identical catalytic activity to the unmodified protein. In summary we conclude that the catalytic activity of PASK is negatively regulated by amino terminal segments of the enzyme, particularly corresponding to the two PAS domains of the enzyme. Likewise the K_(m) for substrate of this enzyme can be modified by phosphorylation of two threonine residues corresponding to the activation loop of the kinase domain.

In summary, biochemical studies of PASK have described an enzyme that is fundamentally very similar to the AMPK and Snf1 enzymes. Like PASK, AMPK activity is repressed by the non-catalytic regulatory domain of the enzyme (reviewed in [4]). When the regulatory domain is derepressed by increased AMP concentration, or is removed by engineered deletion, the catalytic rate of the kinase domain is substantially enhanced [5, 6]. The amino acid sequence of the catalytic domain of PASK is more similar to those of the AMPK/Snf1 and Pim1 enzymes than any other proteins in public databases, and like the AMPK/Snf1 and Pim1 enzymes, this catalytic domain is subject to phosphorylation-mediated activation in its activity for substrate. Moreover, the amino terminal domain of PASK, which contains two PAS domains, negatively regulates the catalytic activity of its serine/threonine kinase domain in cis—as does the regulatory domain of AMPK/Snf1. Finally, the PAS domains of PASK can perform a sensing function.

Genetic studies of PASK in budding yeast. The genome of the budding yeast, Saccharomyces cerevesiae, contains genes encoding two highly related forms of the PASK enzyme. One gene, designated PSK1, is located on the left arm of chromosome 1, corresponding to the open reading frame (ORF) YAL017W. The other, designated PSK2, is located on the left arm of chromosome 15, corresponding to ORF YOL045W. The two forms of yeast PASK are more related in primary amino acid sequence to one another than either are to the enzymes encoded by flies or humans. As such, we tentatively conclude that they arose as a duplication subsequent to the evolutionary divergence between yeast and the other metazoan organisms presently under study. The data indicate, however, that the yeast forms of PASK represent bona fide orthologs of the metazoan enzymes. The amino acid sequence of the catalytic domains of the two yeast enzymes are more highly related to those of the metazoan PASK enzymes than any other protein kinase, including all protein kinases encoded by the genome of S. cerevesiae. The same holds for both PAS domains of the two yeast enzymes. The PAS-A domains of both yeast enzymes are more highly related in primary amino acid sequence to the PAS-A domains of metazoan PASK enzymes than any PAS domain available in public databases. Likewise, the PAS-B domains of the yeast enzymes are more highly related to the PAS-B domains of metazoan PASKs than any other PAS domain. Finally, we have obtained unequivocal evidence that human PASK effectively complements the growth defects of yeast strains lacking both PSK1 and PSK2 enzymes (see below).

The individual yeast PSK1 and PSK2 genes were deleted by homologous recombination. In both cases a targeting vector was prepared to replace the entire open reading frame with a selectable marker. Evidence of successful deletion of each gene was confirmed by Southern blotting, yet no obvious phenotypic effect on growth under normal conditions was observed. A doubly-deleted strain (psk1 psk2) was generated by eliminating the PSK2 gene from a haploid, psk1 mutant strain. Again, no obvious effects on vegetative growth were observed. The doubly-deleted strain was then subjected to a wide range of culture conditions in an effort to identify conditions that might reveal differences between wildtype yeast, singly-mutated variants and the doubly-deficient strain. Among 50 prototypical growth conditions tested [7], two were observed to differentially affect the growth of the strain lacking both PSK1 and PSK2 genes. Cultures containing elevated levels of zinc (10 mM), when incubated at 38° C., supported the growth of wildtype yeast to a more substantive degree than the psk1 psk2 double mutant. Under such conditions the PSK2-deleted strain grew at an intermediate level, whereas the PSK1-deficient strain was similar to wildtype. Very similar observations were made when vegetative growth at 38° C. was monitored on minimal-media culture plates with galactose rather than glucose as the carbon source. Under such conditions, the wildtype strain grew well, psk1 cells grew at a slightly reduced rate, psk2 cells grew at a markedly reduced rate, and the psk1 psk2 double mutant grew very poorly. Under both conditions, reintroduction of PSK2 on a plasmid completely restored growth to wildtype levels.

Having established culture conditions capable of distinguishing vegetative growth rate, efforts were made to identify high-copy suppressors of the psk1 psk2 double mutant phenotype. Two independently generated libraries of yeast genomic DNA, carried on a high-copy plasmid bearing the yeast URA3 gene (pRS426) [8], were transformed into the double mutant. Cells were initially selected on minimal media culture plates supplemented with glucose, yet lacking uracil. Three to four days later Ura⁺ colonies were scraped from each dish, pooled and plated onto minimal media culture plates with galactose and incubated at 38° C. Under such conditions, cells of the psk1 psk2 double mutant fail to grow. When transformed with either of the two genomic libraries, however, evidence of high-copy suppression was observed in the form of healthy colonies. Suppressing plasmids were rescued from individual colonies, transformed into E. coli, isolated and sequenced in order to identify putative high-copy suppressors. This effort led to the isolation and identification of roughly 50 high-copy suppressors of the psk1 psk2 double mutant phenotype. Among these, we focused our attention on clones that were retrieved a minimum of two independent times, amounting to fifteen independent genes (Table 4). Each of these genes was retransformed into psk1 psk2 mutants and the suppressing phenotype was confirmed. Two of the suppressing genes are the PSK genes themselves, demonstrating the validity of the screen. Three of the genes encode proteins involved in sugar metabolism; including phosphoglucose mutase 1 (Pgm1p), phosphoglucose mutase 2 (Pgm2p), and Snf1 interacting protein (Sip1p). Pgm1p and Pgm2p are functionally redundant and represent critical enzymes in the conversion of galactose to glucose [9, 10], and Sip1p is a positive regulator of the Snf1p kinase controlling alternate carbon source utilization [11 ]. A fifth gene repeatedly isolated in the suppressor screen, designated DDP1, encodes diadenosine polyphosphate hydrolase [12, 13].

Among the remaining ten genes that were retrieved at least two independent times in the high-copy suppressor screen, seven—and possibly two others—encode products involved in RNA metabolism or translation. These include: (i) the DED1 and DBP1 genes—both of which encode RNA helicases involved in translation initiation [14-16]; (ii) EDC1—which encodes an enhancer of mRNA decapping [17] (iii) RPR1 and POP4—which respectively encode the RNA and a protein component of RNaseP [18, 19]; (iv) tG(CCC)D which encodes a glycine tRNA [20]; and (v) RDN which encodes the RNA subunits of the ribosome [21]. Additionally, plasmids containing the YDL189W ORF were recovered multiple, independent times in the high-copy suppressor screen. Although no function has been ascribed to this gene, the encoded protein contains an R3H single-stranded nucleic acid binding domain [22], indicating that this gene product may also be involved in RNA metabolism or translation. Finally, the CPA1 gene encoding carbamoyl phosphate synthetase also emerged as a bona fide high-copy suppressor of the psk1 psk2 double mutant phenotype. Although the product of this gene acts at the first catalytic step of arginine biosynthesis [23], Cpa1p translation is tightly regulated via a μ-ORF in its 5′UTR [24, 25].

TABLE 4 High-copy suppressors of psk1 psk2 mutation. Gene # Description PSK1/PSK2 4 PAS Kinase PGM1/PGM2 3 Galactose Metabolism SIP1 5 Glucose Repression DDP1 4 Diadenosine Polyphosphate Hydrolase DED1 3 RNA Helicase DBP1 2 RNA Helicase EDC1 2 Enhancer OF mRNA Decapping RPR1 2 RNase P RNA Subunit POP4 3 RNase P/RNase MRP Subunit tG(CCC)D 2 tRNA Gly RDN 3 Ribosomal RNA subunits YDL189W 10 R3H Domain Protein CPA1 4 ARG Biosynthesis ADE16 2 Adenine Biosynthesis

For this data table, suppressors were recovered multiple times. The first column gives the gene or ORF name. The second gives the number of independent times this gene was recovered in the suppressor screen. The third column gives a brief description of the gene products of the high-copy suppressor genes.

Our genetic studies of the two yeast genes encoding PASK have helped establish a framework for understanding the biological role of this enzyme. Yeast cells without both enzymes are significantly impeded in vegetative growth when the cells are supplied with sub-optimal sources of sugar. Since seven of the eleven relevant high-copy suppressors—and as many as nine—encode products relating to RNA metabolism or translation, we conclude that PASK is involved in protein synthesis. Moving forward with this observation we prepared a dominant negative form of the human PASK enzyme in which a conserved lysine residue located within the ATP-binding region of the catalytic domain was mutated to arginine (designated K1028R). Baculovirus-expressed K1028R is completely inactive with respect to both autophosphorylation and trans-phosphorylation of the AMARA peptide. An expression vector encoding the K1028R variant of human PASK was transfected into cultured Rat-1 cells. Stable transformants were isolated and evaluated by western blotting using anti-hPASK antibodies. Two cell lines were chosen for further study, one expressing an intermediate level of the K1028R variant of PASK (designated KRint), and another expressing a higher level of the inactive enzyme (designated KRhi). The growth rate of the KRint cell line was slightly slower than that of Rat-1 cells that had been transfected with an empty expression vector. The KRhi cells doubled much more slowly, roughly 25% of the growth rate of control cells.

The KRhi cells were also observed to be substantially larger than control cells when viewed by light microscopy. Following this observation, FACS experiments were conducted to measure both the size and DNA content of log phase KRhi, KRint and control cells. The size distribution of KRhi cells was substantially different from both the KRint and control cells. More dramatic differences were observed in measurements of DNA content. KRhi cells display a bi-modal DNA contcorresponding to 2N and 4N the diploid DNA content of control Rat-1 cells. These observations are consistent with images of DAPI-stained cells. The nuclei of KRhi cells are considerably larger than those of control Rat-1 cells. In summary, these studies indicate that high levels of expression of a dominant-negative form of PASK slows the mitotic growth of Rat-1 cells and leads to a concomitant increase in both cell size and DNA content.

Our identification of a link between PASK—implicated in translational regulation by yeast genetics—and cell size control has a striking parallel to results of Thomas and colleagues [26]. By conditionally eliminating the 40S ribosomal protein S6 in adult mouse liver tissue, the Thomas laboratory has provided compelling evidence that lesions in the translation apparatus differentially affect mitotic growth as compared with cell size. Hepatocytes deficient in the S6 protein are unable to divide mitotically subsequent to hepatectomy. By contrast, when experimental animals are starved, these same S6-deficient hepatocytes are able to increase in cell size in response to re-feeding. Indeed, entire liver mass expands similarly in control and S6-deficient tissue in response to re-feeding. The very surprising and compelling observations of Thomas and colleagues reveal a fundamental difference between growth in cell size and mitotic growth in translationally-compromised hepatocytes.

The observations reported by Thomas and colleagues are consistent with emerging evidence that the genes encoding certain translation initiation factors are able to contribute to the transformed phenotype of cultured mammalian cells. The most compelling evidence that dysregulation of translation can foster transformation has come from studies of the 5′ cap-binding factor eIF4E. Overexpression of this translation initiation factor has been shown to directly induce malignant transformation of NIH-3T3 cells [27]. Additional evidence indicating that enhancement of translation initiation can foster the transformed phenotype has come from studies of eIF4E expression levels in human tumors. As reviewed by Clemens and Bommer [28] and DeBendetti and Harris [29], eIF4E levels are significantly elevated in many solid tumors and tumor cell lines. The most pronounced increases in eIF4E expression have been found in breast cancer tissues, and head and neck squamous cell carcinomas [30-34]. Although evidence favoring the role of enhanced translation in the formation of human tumors is strongest in the case of eIF4E, elevated levels of mRNAs encoding eIF4A and eIF4G have also been detected in melanoma and squamous cell lung carcinoma tumors [35, 36]. Finally, the p48 subunit of eIF3 has been identified as the protein encoded by the int-6 gene, which is a site of frequent integration in the mouse genome by mouse mammary tumor virus [37].

Extensive mechanistic studies have resolved the biochemical functions of both positively- and negatively-acting translation initiation factors. The activity of the eIF4E cap binding factor is negatively regulated by proteins designated eIF4E binding proteins (4E-BPs). As reviewed by Raught and Gingras [38], both eIF4E and 4E-BPs are regulated by phosphorylation. The MAP kinase pathway (growth factor regulated), TOR pathway (growth factor regulated) and p38 pathway (stress regulated) have all been implicated in the phosphorylation of 4E-BPs. When phosphorylated, the 4E-BPs are released from eIF4E, allowing the cap binding protein to interact with eIF4G, form a stable interaction with 5′ cap structures, and enhance the efficiency of translation initiation. Most components of this translational regulatory complex have been shown to be cytoplasmic and many tend to be enriched in the perinuclear region of mammalian cells [39]. Notably, PASK is identically distributed as assayed by immunohistochemical staining of cultured mammalian cells. In sum, these data indicate PASK can act by directly regulating the activity of specific translation initiation factors.

Biophysical studies of the PASK enzyme. In parallel with the functional studies of PASK, we undertook NMR-based studies of the structure, dynamics and ligand binding of the PAS domains of this protein. Our goals were two-fold: to determine if these domains are structurally homologous to other members of the PAS domain family and if so, to investigate whether they are capable of binding small molecule ligands in a similar manner.

Given the requirement for large (10-100 mg) quantities of soluble, isotopically-labeled protein for NMR studies, our first step was to optimize expression of PAS-containing constructs in bacterial systems. To this end, we used secondary structure prediction methods [40] to identify PAS domains from the primary amino acid sequence of PASK and design constructs that avoided having termini within secondary structure elements. These were cloned into a series of “parallel” expression vectors optimized for ease of cloning and expression [41] to rapidly generate a group of plasmids that can use bacteriophage T7-based systems to express the PAS fragments as C-terminal fusions to several partner proteins. As SDS-PAGE analysis identified several soluble fragments of PAS-A, we initially concentrated our attention on those.

We evaluated soluble fragments of PAS-A for their suitability for NMR studies by using a rapid NMR-based screening method [42, 43]. This approach is based on 2D ¹⁵N/¹H HSQC spectra of E. coli cell extracts from uniformly ¹⁵N-labeled cultures that have overexpressed proteins of interest. For proteins overexpressed at the levels we typically observe from T7-promoter driven systems, the high abundance of these proteins in the extract ensures that signals in these spectra are exclusively derived from the protein of interest. From these spectra, we can qualitatively estimate the number of residues in random coil conformations from the amide proton chemical shift dispersion, which is typically poor for unfolded proteins (7.8-8.4 ppm) as compared to folded proteins (6-10 ppm). As no protein purification is required, and these spectra can be acquired in 15-60 minutes on a 500 MHz NMR instrument, this method provides a quick assessment of folding and project feasibility at an early stage. Using this approach for a series of C-terminal deletion constructs of the PAS-A domain, we identified residues 131-237 as a minimal folding unit for this domain.

Having identified a well-folded PAS-A fragment that included residues 131-237, we began structural studies of this domain. Using size exclusion chromatography and ¹⁵N relaxation measurements, we established that this fragment was monomeric and thus could use standard triple resonance approaches to obtain ²⁵N, ¹³C and ¹H chemical shift assignments. As several reviews of these methods are available [44, 45], we will briefly survey this approach here. All of the NMR experiments were run on 1 mM samples of uniformly ¹⁵N or ¹⁵N/¹³C labeled PAS-A using 500 and 600 MHz NMR spectrometers. Backbone chemical shift assignments were based on 3D HNCACB, CBCA(CO)NH, HNCO and HNHA spectra. Sidechain assignments for non-aromatic residues were obtained from 3D H(CCO)NH-TOCSY, (H)C(CO)NH-TOCSY and HCCH-TOCSY methods. Stereospecific assignments of Val and Leu methyl groups were based on constant time ¹³C/¹H spectra acquired on a uniformly 10% ¹³C-labeled sample [46]. Proton chemical shift assignments for the ring systems of the nine aromatic amino acids were based on 2D NOESY, DQF-COSY and TOCSY spectra recorded on an unlabeled PAS-A sample in 99.9% D₂O. Using this combination of methods, we obtained virtually complete chemical shift assignments for PAS-A.

Using these assignments, we determined the solution structure of PAS-A from the following information. Distance restraints were obtained from two isotope-edited 3D NOESY experiments (¹⁵N- and ¹⁵N,¹³C-edited [47]; τ_(m)=150 ms) run on a uniformly ¹⁵N¹³C sample in 90% H₂O: 10% D₂O and a 2D NOESY spectrum (τ_(m)=120 ms) of an unlabeled sample in 99.9% D₂O. NOE peak intensities were qualitatively interpreted into three distance classes, accounting for chemical shift degeneracy [48]. Restraints on the backbone dihedral angles φ and ψ were derived from analyses of backbone ¹H, ¹⁵N and ¹³C chemical shifts using the program TALOS [49] with minimum bounds of ±30°. Hydrogen bond restraints were derived from qualitative analyses of ²H exchange data, defining protected amides as those with significant protonation after 1 hour in 99.9% D₂O at pH 6.5, 25° C. Structures were generated with a standard simulated annealing protocol in CNS [50] using 515 manually assigned NOE-based distance restraints, 116 dihedral angle restraints and 48 hydrogen bond restraints. These structures were subsequently refined using an ambiguous NOE protocol as implemented in the ARIA enhancement of CNS [51]. Using ARIA-based interpretation of the 2D and 3D NOE spectra, we increased the number of interproton distance restraints from 515 to 2520. Furthermore, 61 ¹⁵N/¹H residual dipolar coupling restraints were added based on measurements made on a sample of PAS-A in a suspension of 10 mg/mL Pf1 filamentous bacteriophage [52].

We generated a group of 20 structures from this information with low total energy and few violations of experimental restraints. The high precision of these structures is indicated in the low backbone r.m.s. deviation of individual structures to the mean: 0.44±0.08 Å for residues located outside the Fα/FG region. This precision is typical of structures with a high average number of restraints per residue, as we have here (˜25 restraints/residue). Most sidechains within the core of the protein are also well defined, with an r.m.s. deviation of 0.95±0.12 Å. Comparing these structures to those of other PAS domains [53-56] show that all have a similar global α/β fold, with a backbone r.m.s. deviation of 1.2-1.5 Å for residues in the β-sheet and N-terminal Cα, Dα and Eα helices.

Comparison of PAS domain structures reveals a key difference between the PAS-A domain of PASK and other PAS domains. In contrast to the long Fα helix observed in other structures, this helix and the subsequent FG loop are significantly disordered in PASK. This is consistent with our observations that signals from these regions were attenuated or missing in several of the spectra used for backbone chemical shift assignment. To investigate this further, we measured ¹⁵N relaxation rates to characterize the backbone dynamics of PAS-A. ¹⁵N T₁, T₂ and heteronuclear ¹⁵N{¹H} NOE values were determined using standard ¹⁵N/¹H HSQC-based experiments [57] and analyzed with a reduced spectral density function mapping approach [58, 59]. These results provide an experimental measurement of flexibility of the protein backbone on several timescales. Most of the protein is well ordered, as judged by the relatively uniform J values in residues in the Aβ-Dα and Gβ-Iβ secondary structure elements. However, the region between Eα and Gβ shows increased J(0.87Φ_(H)) and J_(eff)(0) values, consistent with increased flexibility on the nanosecond and millisecond timescales. This contrasts with ¹⁵N relaxation measurements on other PAS domains[55], which do not show any unusual flexibility in any secondary structure elements.

Based on our studies to this point, two lines of evidence pointed to the possibility that the PAS-A domain was capable of binding small ligands. First, the combination of structural and dynamic data clearly demonstrates the flexibility of the Fα and FG regions. Both of these regions are intimately involved in PAS/ligand interactions as shown in the structure of the FixL PAS domain bound to heme [56]. If PAS-A binds ligands in an induced fit manner, it is reasonable that these regions will be flexible in the absence of compound. Second, structure-based alignments show that key positions [60] in PAS-A are occupied by amino acids that may allow small molecules to bind in the core of this domain. These same positions are occupied by large hydrophobes in PAS domains that bind small ligands (PYP [53]) or no ligands (HERG [54]), effectively filling the central core. In contrast, FixL has two glycines in these positions to facilitate binding of the large heme ligand [56]. In PASK these sites are taken by amino acids that may similarly allow access into the core of the domain, due to their small size (Cys) or conformational preferences (Ile).

To directly investigate the ligand-binding capability of the PAS-A domain, we used NMR-based methods to screen a small library of synthetic organic compounds. This library consisted of approximately 150 compounds with formula weights between 200-300Da. Compounds were screened by adding groups of 5 (500 μM each) into samples of uniformly ¹⁵N-labeled protein (250 μM) and monitoring ligand-induced conformational changes in PAS-A by recording ¹⁵N/¹H HSQC spectra on these samples. If significant chemical shift changes were observed in these multiligand samples, we deconvoluted the contribution of each by examining the shifts of each ligand individually in new samples. In this manner, we identified eight compounds that bound PAS-A with K_(d) values (measured by NMR) below 1 mM. Spectra showing the chemical shift changes caused by the tightest binding of these compounds were determined. These data show that titrating this compound into a PAS-A sample leads to chemical shift changes in a limited number of residues that cluster in a region that is highly analogous to the heme-binding site of FixL [56].

We have obtained structure/activity relationship (SAR) information by examining the binding characteristics of structurally related compounds. For example, compound #134 has a bibenzyl architecture, a common feature of many protein-binding ligands [61, 62]. Comparing this ligand to other bibenzyl compounds in the library establishes the importance of a hydroxyl group located in the ethylene linker (Table 5). This information is used to guide construction of secondary libraries screened to improve the affinity and specificity of binding to PAS targets.

TABLE 5

Structure-activity information on PAS-A binding compounds. compound R1 R2 K_(d) #134

—H ˜100-200 μM #130

—CH₃ (>5 mM) #133

—H (>5 mM)

Comprehensive biochemical HTS for identifying specific, small molecule activators and inhibitors of PASK. We have adapted PASK protein binding assays, such as kinase and NMR-based assays, to high throughput screening (HTS). A compound collection initially consisting of roughly 350,000 drug-like chemicals has been collected, organized and extensively characterized in over 100 independent HTS assays. Such efforts have enabled the discovery of a large number of chemicals that potently and selectively modulate the activities of a broad range of polypeptide targets. The compound library has produced numerous drug entities that, following extensive optimization by medicinal chemistry, are in various phases of clinical and pre-clinical testing.

PASK HTS kinase assays identify both agonists and antagonists of the catalytic ability of the enzyme to phosphorylate the AMARA peptide. Our evidence indicates that the PASK PAS domains function as molecular sensors, negatively regulating the enzyme under conditions that are susceptible to depression in response to the appropriate intracellular signal. This parallels the AMPK system, which uses a regulatory domain and two negatively acting subunits to repress catalytic activity and simultaneously serve as the sensing device that monitors intracellular AMP/ADP-ATP ratios [4]. PAS domains have been reported to act as biosensors in studies of the aryl hydrocarbon receptor [73], photoactive yellow protein of the phototrophic bacterium E. halophila [74] and the bacterial Aer [75] and FixL [76] proteins.

One HTS assay kinase assay we employ is a robust and simple, non-radioactive chemiluminescent assay that measures the ability of the human enzyme to phosphorylate a biotinylated derivative of the AMARA peptide. The enzyme is expressed in baculovirus and purified by affinity and ion-exchange chromatography under routine conditions. Assay buffer is composed of 20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 2 mM EGTA, 0.1% NP-40, 0.5 mM benzamidine, 1 mM NaF and 10 mM DTT. Enzyme is added to this buffer at a concentration determined to be in the linear range of activity (long per microtiter plate well). For the most robust and sensitive kinase assay, we use an ATP concentration matching the K_(m) for PASK (2.5 mM). After dispensing 80 μl of the enzyme/buffer mix per well in Costar 96-well microtiter plates (coated with Neutravidin at 0.5 mg/ml at room temperature for 2 hr), 10 μl of test compounds is added at a final concentration of 1 μM compound. Compounds are diluted in DMSO from master plates stored in DMSO at 10 mM. Following compound addition, the reactions are initiated by addition of 10 μl of biotinylated AMARA peptide and ATP such that the final concentration of substrate and ATP are 1 mM and 2.5 mM respectively. Instrumentation for the addition of buffer/enzyme mix, diluted compounds and substrate/ATP is the Robbins Hydra 96 multipipette. Following brief shaking on a microtiter plate shaker (Lab-Line Instruments), plates are incubated for 1 hr at room temperature, washed 3× with distilled H₂O using a LABTEK 96-well wash, and applied with 100 μl per well of antibody buffer. The primary antibody is a mouse monoclonal antibody specific to the phosphorylated AMARA peptide (Pan Vera). The secondary antibody is an HRP-labeled rabbit, anti-mouse antibody (New England Biolabs). The final antibody buffer mix contains 2% BSA in PBS. After antibody addition the plates are briefly shaken, incubated at room temperature for 1 hr and washed 3× with H₂O. Reactions are developed by addition of 100 μl of Super Signal substrate (Pierce) and read calorimetrically using a Wallac Victor2, 1420 Multilabel Counter. Target specificity is confirmed by comparative assays with control non-PASK protein kinases.

Compounds showing good dose response curves, corresponding to potent agonistic or antagonistic activity, are tested against six versions of the human PASK enzyme. Three of the enzyme samples represent the intact protein in: (i) its native state as purified from baculovirus; (ii) its activated state following autophosphorylation; (iii) and its inactivated state following treatment with lambda phosphatase. Concomitant experiments are conducted using the amino-terminal truncated enzyme lacking its two PAS domains, again using the native enzyme as well as the phosphorylated and dephosphorylated derivatives. This secondary level of follow-up distinguishes between compounds that act on the kinase domain versus those that require the PAS domains for either stimulatory or inhibitory activity. Compounds from the HTS screen are evaluated by HPLC, mass spectrometry and NMR spectroscopy, not only of the starting compound, but also of analogs produced as a means of establishing a structure activity relationship (SAR) between the compound and its ability to either inhibit or stimulate PASK.

High throughput NMR-based protein/ligand screening. Two important facets of NMR-based protein/ligand screens are the size and scope of the compound libraries that are being used. One type of library we use is a “directed” library, consisting of a small (100-200) number of compounds whose structures are based on common structural elements of PAS-binding ligands, including biological cofactors (heme, NADH, FMN, FADH₂), chromophores (hydroxycinnaminic acid), environmental toxins (tetrachlorodibenzodioxin [TCDD]), etc. These have several features in common, including planarity, conjugation and limited charge at neutral pH. Chemical databases are searched with these criteria, starting with substructures that match common frameworks of PAS ligands. Additional geometric criteria are obtained from observations that protein-binding ligands are inherently biased towards certain chemical architectures [61, 79]. Many of these architectures are aromatic-rich and include the general structures of several PAS-binding ligands including PASK-binding compound #134 (Table 5). We also screen larger libraries (1000-2000 compounds) that are designed to cover a wider range of chemical structures while still taking advantage of the observed biases of protein-binding ligands [61, 79]. Compounds in this library are also chosen with an emphasis towards later use in synthetic approaches, with relatively low formula weight (100-200Da) and composition of functional groups.

A wide variety of NMR-based methods are available to rapidly screen libraries of small compounds for binding to protein targets [82]. We primarily use protein-based screening methods, which are well suited for PAS domains given their small size (10-15 kDa) and the structural information obtained from the studies outlined above. As demonstrated with PAS-A, we screen using isotopically labeled protein and unlabeled ligands, using HSQC-type experiments to selectively observe protein signals in the presence of excess ligand. Compounds are maintained as 1M stocks in deuterated DMSO, and protein samples checked to ensure that DMSO does not bind with any significant affinity. Samples of ¹⁵N/¹³C-methyl labeled protein (250 μM) are mixed with 3-10 compounds at 1 mM each, which is sufficient to find ligands with weak (millimolar) dissociation constants. Protein chemical shifts are recorded using ¹³C/¹H HSQC as our primary method, complemented by ¹⁵N/¹H HSQC. Spectra from ligand-containing solutions are compared to those from ligand-free samples, calculating chemical shift changes with the minimum chemical shift difference method [83]. Where significant changes are observed, we deconvolute the binding of each ligand in the mixture by examining new samples with single protein/ligand mixtures. Compounds that demonstrate binding are titrated into a sample of ¹⁵N-labeled protein to measure dissociation constants, which can be measured by NMR if the complex is in fast exchange (K_(d)>10 μM).

Screening throughput of this method can exceed 1000 compounds per day for the initial screen: 15 minutes per sample (10′ experimental, 5′ sample manipulation) and 10 ligands/sample. PAS-binding compounds identified in this screen are evaluated for their ability to modulate PASK function in vitro using the previously described peptide phosphorylation assay. Compounds are checked for effects on both full length and truncated versions of PASK containing only the kinase domain to ensure that any modulatory action is mediated through the PAS domains.

We use several sources of structure-activity relationship information available from the first round of screening to generate small secondary libraries that we screen to find compounds with higher affinity. As shown above, comparisons of the affinity of structurally-related compounds identify positions on ligands that are essential for binding. Additionally, comparisons of the chemical shift changes caused by related ligands rapidly identify the relative orientation of protein/ligand complexes [85], potentially identifying sites on these ligands that may be amenable to modification as an avenue to increase affinity. The combination of these approaches allows us to design libraries that are simple modifications of ligands from the first screen.

An alternative approach that generates more drastically altered ligands for the second library is the SAR-by-NMR method [86]. We conduct two screens through the primary library with the goal of finding two ligands that bind at adjacent sites on the protein structure. This is achieved by a first screen as previously discussed, followed by a second screen through the library in the presence of saturating concentrations of a ligand identified by the first screen. Through the use of protein-directed NMR experiments such as the ¹³C/¹H HSQC, one can readily identify compounds from the second screen that bind at an adjacent site to the first compound. We then obtain a structure of the ternary complex of the protein bound to both ligands. This is achieved by obtaining similar structural information as used for the PAS-A structure determination complemented by intermolecular distance restraints using isotope-filtered, edited 3D NOE experiments [87] that provide the selective observation of NOEs between isotopically-enriched proteins and non-enriched ligands. Guided by this structure, we identify sites on each ligand that can be used to generate a linker between the two compounds in the expectation that the linked compound will have enhanced affinity over the two separate compounds. Libraries are constructed from this information using a series of linkers with variable length and flexibility. We screen these secondary library compounds for binding to the PAS-A and PAS-B domains as described above.

Phosphorylation Screening. An alternative strategy to identify PASK substrates follows methods developed by Hunter and colleagues for the identification of protein kinase substrates by expression screening with solid-phase phosphorylation. This method, termed “phosphorylation screening,” represents an adaptation of classical methods of expression screening using bacteriophage lambda. Hunter and colleagues have used this approach to identify known and novel substrates for the Erk1 [65] and Cdk2 [66] protein kinases.

Yeast cDNA are cloned into the λGEX5 vector developed by Hunter and colleagues. This bacteriophage lambda vector contains a plasmid sequence between two NotI sites, consisting of a ColE1 origin, the ampicillin resistance gene, and a GST gene followed by a small “stuffer” sequence to be replaced by cDNA inserts. Compared with the original λgt11 vector, the λGEX5 vector provides three important advantages. First, clones encoding polypeptides phosphorylated by PASK can be rapidly converted into plasmid clones by excision rescue without purifying cDNA fragments. Second, rescued plasmids can be directly utilized not only for cDNA sequencing but also for expression of GST fusion proteins for further characterization. Finally, GST, the N-terminal fusion partner of the recombinant product expressed by λGEX5, is highly soluble, easy to purify by GSH-agarose chromatography, and much smaller than the β-galactosidase component of the fusion proteins expressed by λgt11.

Expression libraries are screened according to the methods of Hunter and Fukunaga [65] using human PASK expressed in baculovirus. Prior to the phosphorylation step of the assay, filter lifts are incubated in the presence of unlabelled ATP as a means of reducing the frequency of isolating clones whose products have either autophosphorylating or ATP-binding activity. Highly purified, auto-phosphorylated (activated) human PASK is added with [γ-³²P]ATP in optimal buffer conditions. Following incubation, the filters are washed and exposed to X-ray film to identify plaques expressing polypeptide substrates for PASK. Optimization strategies outlined by Hunter and Fukunaga are followed to expedite completion of a successful screen. After retrieving nitrocelluose lifts bearing lambda-expressed proteins, filters are exposed to 6M urea followed by sequential dilution and ultimate removal of the urea. This approach helps solubilize over-expressed proteins which are often restricted to inclusion bodies. In the case of expression cloning of eukaryotic transcription factors, inclusion of the urea solubilization step substantially enhanced signal to noise ratios [67].

Positive plaques are isolated, from which encoding plasmids are excised and sequenced. Particularly interesting substrate proteins are further studied to identify precise sites of phosphorylation using GST-fusion proteins expressed and purified from E. coli. Finally, individual serine and threonine sites are altered by site directed mutagenesis followed by reintroduction of the mutant on a plasmid in a strain deleted for the chromosomal copy of the gene so as to facilitate further biochemical and biological assays.

In addition to phosphorylation screening, we employ an entirely different approach described by Kirschner and colleagues [68-70]. This approach also relies on the screening of cDNA libraries, yet in this case the assay is conducted on pools of in vitro translation products. In brief, a cDNA library is prepared from poly(A)+ yeast RNA cloned into the pCS2+ vector [71]. cDNAs larger than 0.5 kb is ligated directionally into pCS2+ and electroporated into competent E. coli cells. The pCS2+ plasmid contains regulatory sequences for coupled transcription/translation immediately upstream from the directionally cloned cDNA insert site, thus facilitating TNT-mediated transcription/translation in reticulocyte lysates. We follow the methods of Kirshner and colleagues to simultaneously at express pools of ˜100 cDNA clones, typically resulting in the synthesis of 15-30 ³⁵S-labeled polypeptides. These pools of in vitro translated protein will subsequently be incubated with baculovirus-expressed PASK under optimal conditions for its enzymatic activity. Following phosphorylation with cold ATP, the ³⁵S-labeled protein pools are run on SDS-PAGE gels followed by autoradiographic imaging. Control reactions are conducted in the absence of PASK such that pooled samples can be compared side-by-side on SDS-PAGE gels. Proteins phosphorylated by PASK are provisionally identified by a shift in their electrophoretic mobility. Pools containing cDNA capable of expressing PAS-kinase modified polypeptides are de-convoluted in order to identify relevant cDNAs and their encoded polypeptides.

Protocol for high throughput PASK autophosphorylation assay.

A. Reagents

Neutralite Avidin: 20 μg/ml in PBS.

kinase: 10⁻⁸−10⁵M PASK kinase domain at 20 μg/ml in PBS.

substrate: 10⁻⁷−10⁻⁴M biotinylated PASK substrate at 40 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.

Assay Buffer: 100 mM KCl, 10 mM MgCl₂, 1 mM MnCl₂, 20 mM HEPES pH 7.4, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktail of protease inhibitors.

[³²P]μ−ATP 10× stock: 2×10⁻⁵M cold ATP with 100 μCi [³²P]μ-ATP. Place in the 4° C. microfridge during screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVo₃ (Sigma #S-6508) in 10 ml of PBS.

B. Preparation of Assay Plates

Coat with 120 1 μl of stock N Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay

Add 40 μl assay buffer/well.

Add 40 μl biotinylated substrate (2-200 pmoles/40 ul in assay buffer)

Add 40 μl kinase (0.1-10 pmoles/40 ul in assay buffer)

Add 10 μl compound or extract.

Add 10 μl [³²P]γ-ATP 10×stock.

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Stop the reaction by washing 4 times with 200 μl PBS.

Add 150 μl scintillation cocktail.

Count in Topcount.

D. Controls for All Assays (Located on Each Plate)

a. Non-specific binding

b. cold ATP at 80% inhibition.

Protocol for High Throughput PASK PAS Domain—ligand Binding Assay.

A. Reagents

Neutralite Avidin: 20 μg/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl₂, 1% glycerol, 0.5% NP-40, 50 mM b-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors.

³³P PASK polypeptide 10×stock: 10⁻⁸−10⁻⁶M “cold” PASK PAS domain supplemented with 200,000-250,000 cpm of labeled PASK (Beckman counter). Place in the 4° C. microfridge during screening.

Protease inhibitor cocktail (1000×): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVO₃ (Sigma #S-6508) in 10 ml of PBS.

Ligand: 10⁻⁷−10⁻⁵M biotinylated ligand in PBS.

B. Preparation of Assay Plates

Coat with 120 μl of stock N-Avidin per well overnight at 4° C.

Wash 2 times with 200 μl PBS.

Block with 150 μl of blocking buffer.

Wash 2 times with 200 μl PBS.

C. Assay

Add 40 μl assay buffer/well.

Add 10 μl compound or extract.

Add 10 μl ³³P-PASK polypeptide (20-25,000 cpm/0.1-10 pmoles/well=10⁻⁹−10⁻⁷ M final conc).

Shake at 25° C. for 15 minutes.

Incubate additional 45 minutes at 25° C.

Add 40 μM biotinylated ligand (0.1-10 pmoles/40 μl in assay buffer)

Incubate 1 hour at room temperature.

Stop the reaction by washing 4 times with 200 μM PBS.

Add 150 μM scintillation cocktail.

Count in Topcount.

D. Controls for All Assays (Located on Each Plate)

a. Non-specific binding

b. Soluble (non-biotinylated ligand) at 80% inhibition.

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All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

9 1 3972 DNA human CDS (1)..(3969) 1 atg gag gac ggg ggc tta aca gcc ttt gaa gag gac cag aga tgc ctt 48 Met Glu Asp Gly Gly Leu Thr Ala Phe Glu Glu Asp Gln Arg Cys Leu 1 5 10 15 tcc cag agc ctc ccc ttg cca gtg tca gca gag ggc cca gct gca cag 96 Ser Gln Ser Leu Pro Leu Pro Val Ser Ala Glu Gly Pro Ala Ala Gln 20 25 30 acc act gct gag ccc agc agg tcg ttt tcc tca gcc cac aga cac ctg 144 Thr Thr Ala Glu Pro Ser Arg Ser Phe Ser Ser Ala His Arg His Leu 35 40 45 agc aga agg aat ggg ctt tcc aga ctc tgc cag agc agg aca gcg ctc 192 Ser Arg Arg Asn Gly Leu Ser Arg Leu Cys Gln Ser Arg Thr Ala Leu 50 55 60 tct gaa gac aga tgg agc tcc tat tgt cta tca tca ctg gct gcc cag 240 Ser Glu Asp Arg Trp Ser Ser Tyr Cys Leu Ser Ser Leu Ala Ala Gln 65 70 75 80 aat att tgt aca agt aaa ctg cac tgc cct gct gcc cct gag cac acg 288 Asn Ile Cys Thr Ser Lys Leu His Cys Pro Ala Ala Pro Glu His Thr 85 90 95 gac ccg tcc gaa ccg cgg ggc agt gtg tcc tgc tgc tcc ctg ctg cgg 336 Asp Pro Ser Glu Pro Arg Gly Ser Val Ser Cys Cys Ser Leu Leu Arg 100 105 110 gga ctg tcc tca ggg tgg tcc tca cct ctg ctt ccg gcc cct gtg tgc 384 Gly Leu Ser Ser Gly Trp Ser Ser Pro Leu Leu Pro Ala Pro Val Cys 115 120 125 aac cct aac aag gcc atc ttc acg gtg gat gcc aag acc aca gag atc 432 Asn Pro Asn Lys Ala Ile Phe Thr Val Asp Ala Lys Thr Thr Glu Ile 130 135 140 ctg gtt gct aac gac aaa gct tgc ggg ctc ctg ggg tac agc agc cag 480 Leu Val Ala Asn Asp Lys Ala Cys Gly Leu Leu Gly Tyr Ser Ser Gln 145 150 155 160 gac ctg att ggc cag aag ctc acg cag ttc ttt ctg agg tca gat tct 528 Asp Leu Ile Gly Gln Lys Leu Thr Gln Phe Phe Leu Arg Ser Asp Ser 165 170 175 gat gtg gtg gag gcc ctc agc gag gag cac atg gag gcc gac ggc cac 576 Asp Val Val Glu Ala Leu Ser Glu Glu His Met Glu Ala Asp Gly His 180 185 190 gct gcg gtg gtg ttt ggc acg gtg gtg gac atc atc agc cgt agt ggg 624 Ala Ala Val Val Phe Gly Thr Val Val Asp Ile Ile Ser Arg Ser Gly 195 200 205 gag aag att cca gtg tct gtg tgg atg aag agg atg cgg cag gag cgc 672 Glu Lys Ile Pro Val Ser Val Trp Met Lys Arg Met Arg Gln Glu Arg 210 215 220 cgc cta tgc tgc gtg gtg gtc ctg gag ccc gtg gag agg gtc tcg acc 720 Arg Leu Cys Cys Val Val Val Leu Glu Pro Val Glu Arg Val Ser Thr 225 230 235 240 tgg gtc gct ttc cag agc gat ggc acc gtc acg tca tgt gac agt ctc 768 Trp Val Ala Phe Gln Ser Asp Gly Thr Val Thr Ser Cys Asp Ser Leu 245 250 255 ttt gct cat ctt cac ggg tac gtg tct ggg gag gac gtg gct ggg cag 816 Phe Ala His Leu His Gly Tyr Val Ser Gly Glu Asp Val Ala Gly Gln 260 265 270 cat atc aca gac ctg atc cct tct gtg cag ctc cct cct tct ggc cag 864 His Ile Thr Asp Leu Ile Pro Ser Val Gln Leu Pro Pro Ser Gly Gln 275 280 285 cac atc cca aag aat ctc aag att cag agg tct gtt gga aga gcc agg 912 His Ile Pro Lys Asn Leu Lys Ile Gln Arg Ser Val Gly Arg Ala Arg 290 295 300 gac ggt acc acc ttc cct ctg agc tta aag ctg aaa tcc caa ccc agc 960 Asp Gly Thr Thr Phe Pro Leu Ser Leu Lys Leu Lys Ser Gln Pro Ser 305 310 315 320 agc gag gag gcg acc acc ggt gag gcg gcc cct gtg agc ggc tac cgg 1008 Ser Glu Glu Ala Thr Thr Gly Glu Ala Ala Pro Val Ser Gly Tyr Arg 325 330 335 gca tct gtc tgg gtg ttc tgc acc atc agt ggc ctc atc acc ctc ctg 1056 Ala Ser Val Trp Val Phe Cys Thr Ile Ser Gly Leu Ile Thr Leu Leu 340 345 350 ccg gat ggg acc atc cac ggc atc aac cac agc ttc gcg ctg aca ctg 1104 Pro Asp Gly Thr Ile His Gly Ile Asn His Ser Phe Ala Leu Thr Leu 355 360 365 ttt ggt tac gga aag acg gag ctc ctg ggc aag aat atc act ttc ctg 1152 Phe Gly Tyr Gly Lys Thr Glu Leu Leu Gly Lys Asn Ile Thr Phe Leu 370 375 380 att cct ggt ttc tac agc tac atg gac ctt gcg tac aac agc tca tta 1200 Ile Pro Gly Phe Tyr Ser Tyr Met Asp Leu Ala Tyr Asn Ser Ser Leu 385 390 395 400 cag ctc cca gac ctg gcc agc tgc ctg gac gtc ggc aat gag agt ggg 1248 Gln Leu Pro Asp Leu Ala Ser Cys Leu Asp Val Gly Asn Glu Ser Gly 405 410 415 tgt ggg gag aga acc ttg gac ccg tgg cag ggc cag gac cca gct gag 1296 Cys Gly Glu Arg Thr Leu Asp Pro Trp Gln Gly Gln Asp Pro Ala Glu 420 425 430 ggg ggc cag gat cca agg att aat gtc gtg ctt gct ggt ggc cac gtt 1344 Gly Gly Gln Asp Pro Arg Ile Asn Val Val Leu Ala Gly Gly His Val 435 440 445 gtg ccc cga gat gag atc cgg aag ctg atg gaa agc caa gac atc ttc 1392 Val Pro Arg Asp Glu Ile Arg Lys Leu Met Glu Ser Gln Asp Ile Phe 450 455 460 acc ggg act cag act gag ctg att gct gga ggc cag ctc ctt tcc tgc 1440 Thr Gly Thr Gln Thr Glu Leu Ile Ala Gly Gly Gln Leu Leu Ser Cys 465 470 475 480 ctc tca cct cag cct gct cca ggg gtg gac aat gtc cca gaa gga agc 1488 Leu Ser Pro Gln Pro Ala Pro Gly Val Asp Asn Val Pro Glu Gly Ser 485 490 495 ctg cca gtg cac ggt gaa cag gcg ctg ccc aag gac cag caa atc act 1536 Leu Pro Val His Gly Glu Gln Ala Leu Pro Lys Asp Gln Gln Ile Thr 500 505 510 gcc ttg ggg aga gag gaa cct gtg gca ata gag agc ccc gga cag gat 1584 Ala Leu Gly Arg Glu Glu Pro Val Ala Ile Glu Ser Pro Gly Gln Asp 515 520 525 ctt ctg gga gaa agc agg tct gaa cca gtg gat gtg aag cca ttt gct 1632 Leu Leu Gly Glu Ser Arg Ser Glu Pro Val Asp Val Lys Pro Phe Ala 530 535 540 tcc tgc gaa gat tct gaa gct cca gtc cca gct gag gat ggg ggc agt 1680 Ser Cys Glu Asp Ser Glu Ala Pro Val Pro Ala Glu Asp Gly Gly Ser 545 550 555 560 gat gct ggc atg tgt ggc ctg tgt cag aag gcc cag cta gag cgg atg 1728 Asp Ala Gly Met Cys Gly Leu Cys Gln Lys Ala Gln Leu Glu Arg Met 565 570 575 gga gtc agt ggt ccc agc ggt tca gac ctt tgg gct ggg gct gcc gtg 1776 Gly Val Ser Gly Pro Ser Gly Ser Asp Leu Trp Ala Gly Ala Ala Val 580 585 590 gcc aag ccc cag gcc aag ggt cag ctg gcg ggg ggc agc ctc ctg atg 1824 Ala Lys Pro Gln Ala Lys Gly Gln Leu Ala Gly Gly Ser Leu Leu Met 595 600 605 cac tgc cct tgc tat ggg agt gaa tgg ggc ttg tgg tgg cga agc cag 1872 His Cys Pro Cys Tyr Gly Ser Glu Trp Gly Leu Trp Trp Arg Ser Gln 610 615 620 gac ttg gcc ccc agc ccc tct ggg atg gca ggc ctc tcg ttt ggg aca 1920 Asp Leu Ala Pro Ser Pro Ser Gly Met Ala Gly Leu Ser Phe Gly Thr 625 630 635 640 cct act cta gat gag ccg tgg ctg gga gtg gaa aac gac cga gaa gag 1968 Pro Thr Leu Asp Glu Pro Trp Leu Gly Val Glu Asn Asp Arg Glu Glu 645 650 655 ctg cag acc tgc ttg att aag gag cag ctg tcc cag ttg agc ctt gca 2016 Leu Gln Thr Cys Leu Ile Lys Glu Gln Leu Ser Gln Leu Ser Leu Ala 660 665 670 gga gcc ctg gat gtc ccc cac gcc gaa ctc gtt ccg aca gag tgc cag 2064 Gly Ala Leu Asp Val Pro His Ala Glu Leu Val Pro Thr Glu Cys Gln 675 680 685 gct gtc acc gct cct gtg tcg tcc tgc gat ctg gga ggc aga gac ctg 2112 Ala Val Thr Ala Pro Val Ser Ser Cys Asp Leu Gly Gly Arg Asp Leu 690 695 700 tgc ggt ggc tgc acg ggc agc tcc tca gcc tgc tat gcc ttg gcc acg 2160 Cys Gly Gly Cys Thr Gly Ser Ser Ser Ala Cys Tyr Ala Leu Ala Thr 705 710 715 720 gac ctc cct ggg ggc ctg gaa gca gtg gag gcc cag gag gtt gat gtg 2208 Asp Leu Pro Gly Gly Leu Glu Ala Val Glu Ala Gln Glu Val Asp Val 725 730 735 aat tcg ttt tcc tgg aac ctc aag gaa ctc ttt ttc agt gac cag aca 2256 Asn Ser Phe Ser Trp Asn Leu Lys Glu Leu Phe Phe Ser Asp Gln Thr 740 745 750 gac caa acg tca tca aat tgt tcc tgt gct acg tct gaa ctc aga gag 2304 Asp Gln Thr Ser Ser Asn Cys Ser Cys Ala Thr Ser Glu Leu Arg Glu 755 760 765 aca ccc tct tcc ttg gca gtg ggc tcc gat cca gat gta ggc agt ctc 2352 Thr Pro Ser Ser Leu Ala Val Gly Ser Asp Pro Asp Val Gly Ser Leu 770 775 780 cag gaa cag ggg tcg tgt gtc ctg gat gac agg gag ctg tta cta ctg 2400 Gln Glu Gln Gly Ser Cys Val Leu Asp Asp Arg Glu Leu Leu Leu Leu 785 790 795 800 acc ggc acc tgt gtt gac ctt ggc caa ggc cga cgg ttc cgg gag agc 2448 Thr Gly Thr Cys Val Asp Leu Gly Gln Gly Arg Arg Phe Arg Glu Ser 805 810 815 tgt gtg gga cat gat cca aca gaa ccg ctt gag gtt tgt ttg gtg tcc 2496 Cys Val Gly His Asp Pro Thr Glu Pro Leu Glu Val Cys Leu Val Ser 820 825 830 tct gag cat tat gca gca agc gac aga gaa agc ccg gga cac gtt cct 2544 Ser Glu His Tyr Ala Ala Ser Asp Arg Glu Ser Pro Gly His Val Pro 835 840 845 tcc acg ttg gat gct ggc cct gag gac acg tgc cca tca gca gag gag 2592 Ser Thr Leu Asp Ala Gly Pro Glu Asp Thr Cys Pro Ser Ala Glu Glu 850 855 860 cca agg ctg aac gtc cag gtc acc tcc acg ccc gtg atc gtg atg cgc 2640 Pro Arg Leu Asn Val Gln Val Thr Ser Thr Pro Val Ile Val Met Arg 865 870 875 880 ggg gct gct ggc ctg cag cgg gag atc cag gag ggt gcc tac tcc ggg 2688 Gly Ala Ala Gly Leu Gln Arg Glu Ile Gln Glu Gly Ala Tyr Ser Gly 885 890 895 agc tgc cac cat cga gat ggc tta cgg ctg agt ata cag ttt gag gtg 2736 Ser Cys His His Arg Asp Gly Leu Arg Leu Ser Ile Gln Phe Glu Val 900 905 910 agg cgg gtg gag ctc cag ggc ccc aca cct ctg ttc tgc tgc tgg ctg 2784 Arg Arg Val Glu Leu Gln Gly Pro Thr Pro Leu Phe Cys Cys Trp Leu 915 920 925 gtg aaa gac ctc ctc cac agc caa cgc gac tca gcc gcc agg acc cgc 2832 Val Lys Asp Leu Leu His Ser Gln Arg Asp Ser Ala Ala Arg Thr Arg 930 935 940 ctg ttc ctt gcc agc ctg ccc ggc tcc acc cac tct acc gct gct gag 2880 Leu Phe Leu Ala Ser Leu Pro Gly Ser Thr His Ser Thr Ala Ala Glu 945 950 955 960 ctc acc gga ccc agc ctg gtg gaa gtg ctc aga gcc aga ccc tgg ttt 2928 Leu Thr Gly Pro Ser Leu Val Glu Val Leu Arg Ala Arg Pro Trp Phe 965 970 975 gag gag ccc ccc aag gct gtg gaa ctg gag ggg ttg gcg gcc tgt gag 2976 Glu Glu Pro Pro Lys Ala Val Glu Leu Glu Gly Leu Ala Ala Cys Glu 980 985 990 ggc gag tac tcc caa aag tac agt acc atg agc ccg ctg ggc agt ggg 3024 Gly Glu Tyr Ser Gln Lys Tyr Ser Thr Met Ser Pro Leu Gly Ser Gly 995 1000 1005 gcc ttc ggc ttc gtg tgg act gct gtg gac aaa gaa aaa aac aag 3069 Ala Phe Gly Phe Val Trp Thr Ala Val Asp Lys Glu Lys Asn Lys 1010 1015 1020 gag gtg gtg gtg aag ttt att aag aag gag aag gtc ttg gag gat 3114 Glu Val Val Val Lys Phe Ile Lys Lys Glu Lys Val Leu Glu Asp 1025 1030 1035 tgt tgg att gag gat ccc aaa ctt ggg aaa gtt act tta gag atc 3159 Cys Trp Ile Glu Asp Pro Lys Leu Gly Lys Val Thr Leu Glu Ile 1040 1045 1050 gca att cta tcc agg gtg gag cac gcc aat atc atc aag gta ttg 3204 Ala Ile Leu Ser Arg Val Glu His Ala Asn Ile Ile Lys Val Leu 1055 1060 1065 gat ata ttt gaa aac caa ggg ttc ttc cag ctt gtg atg gag aag 3249 Asp Ile Phe Glu Asn Gln Gly Phe Phe Gln Leu Val Met Glu Lys 1070 1075 1080 cac ggc tcc ggc cta gac ctc ttc gct ttc atc gac cgc cac ccc 3294 His Gly Ser Gly Leu Asp Leu Phe Ala Phe Ile Asp Arg His Pro 1085 1090 1095 agg ctg gat gag ccc ctg gcg agc tac atc ttc cga caa cta gtg 3339 Arg Leu Asp Glu Pro Leu Ala Ser Tyr Ile Phe Arg Gln Leu Val 1100 1105 1110 tca gca gtg gga tac ctg cgc ttg aag gac atc atc cac cgt gac 3384 Ser Ala Val Gly Tyr Leu Arg Leu Lys Asp Ile Ile His Arg Asp 1115 1120 1125 atc aag gat gag aac atc gtg atc gct gag gac ttc aca atc aag 3429 Ile Lys Asp Glu Asn Ile Val Ile Ala Glu Asp Phe Thr Ile Lys 1130 1135 1140 ctg ata gac ttt ggc tcg gcc gcc tac ttg gaa agg gga aaa tta 3474 Leu Ile Asp Phe Gly Ser Ala Ala Tyr Leu Glu Arg Gly Lys Leu 1145 1150 1155 ttt tat act ttt tgt ggg acc atc gag tac tgt gca ccg gaa gtt 3519 Phe Tyr Thr Phe Cys Gly Thr Ile Glu Tyr Cys Ala Pro Glu Val 1160 1165 1170 ctc atg ggg aat ccc tac aga ggg ccg gag ctg gag atg tgg tct 3564 Leu Met Gly Asn Pro Tyr Arg Gly Pro Glu Leu Glu Met Trp Ser 1175 1180 1185 ctg gga gtc act ctg tac acg ctg gtc ttt gag gag aac ccc ttc 3609 Leu Gly Val Thr Leu Tyr Thr Leu Val Phe Glu Glu Asn Pro Phe 1190 1195 1200 tgt gag ctg gag gag acc gtg gag gct gcc ata cac ccg cca tac 3654 Cys Glu Leu Glu Glu Thr Val Glu Ala Ala Ile His Pro Pro Tyr 1205 1210 1215 ctg gtg tcc aaa gaa ctc atg agc ctt gtg tct ggg ctg ctg cag 3699 Leu Val Ser Lys Glu Leu Met Ser Leu Val Ser Gly Leu Leu Gln 1220 1225 1230 cca gtc cct gag aga cgc acc acc ttg gag aag ctg gtg aca gac 3744 Pro Val Pro Glu Arg Arg Thr Thr Leu Glu Lys Leu Val Thr Asp 1235 1240 1245 ccg tgg gta aca cag cct gtg aat ctt gct gac tat aca tgg gaa 3789 Pro Trp Val Thr Gln Pro Val Asn Leu Ala Asp Tyr Thr Trp Glu 1250 1255 1260 gag gtg tgt cga gta aac aag cca gaa agt gga gtt ctg tcc gct 3834 Glu Val Cys Arg Val Asn Lys Pro Glu Ser Gly Val Leu Ser Ala 1265 1270 1275 gcg agc ctg gag atg ggg aac agg agc ctg agt gat gtg gcc cag 3879 Ala Ser Leu Glu Met Gly Asn Arg Ser Leu Ser Asp Val Ala Gln 1280 1285 1290 gct cag gag ctt tgt ggg ggc ccc gtt cca ggc gag gct cct aat 3924 Ala Gln Glu Leu Cys Gly Gly Pro Val Pro Gly Glu Ala Pro Asn 1295 1300 1305 ggc caa ggc tgt ttg cat ccc ggg gat ccc cgt ctg ctg acc agc 3969 Gly Gln Gly Cys Leu His Pro Gly Asp Pro Arg Leu Leu Thr Ser 1310 1315 1320 taa 3972 2 1323 PRT human 2 Met Glu Asp Gly Gly Leu Thr Ala Phe Glu Glu Asp Gln Arg Cys Leu 1 5 10 15 Ser Gln Ser Leu Pro Leu Pro Val Ser Ala Glu Gly Pro Ala Ala Gln 20 25 30 Thr Thr Ala Glu Pro Ser Arg Ser Phe Ser Ser Ala His Arg His Leu 35 40 45 Ser Arg Arg Asn Gly Leu Ser Arg Leu Cys Gln Ser Arg Thr Ala Leu 50 55 60 Ser Glu Asp Arg Trp Ser Ser Tyr Cys Leu Ser Ser Leu Ala Ala Gln 65 70 75 80 Asn Ile Cys Thr Ser Lys Leu His Cys Pro Ala Ala Pro Glu His Thr 85 90 95 Asp Pro Ser Glu Pro Arg Gly Ser Val Ser Cys Cys Ser Leu Leu Arg 100 105 110 Gly Leu Ser Ser Gly Trp Ser Ser Pro Leu Leu Pro Ala Pro Val Cys 115 120 125 Asn Pro Asn Lys Ala Ile Phe Thr Val Asp Ala Lys Thr Thr Glu Ile 130 135 140 Leu Val Ala Asn Asp Lys Ala Cys Gly Leu Leu Gly Tyr Ser Ser Gln 145 150 155 160 Asp Leu Ile Gly Gln Lys Leu Thr Gln Phe Phe Leu Arg Ser Asp Ser 165 170 175 Asp Val Val Glu Ala Leu Ser Glu Glu His Met Glu Ala Asp Gly His 180 185 190 Ala Ala Val Val Phe Gly Thr Val Val Asp Ile Ile Ser Arg Ser Gly 195 200 205 Glu Lys Ile Pro Val Ser Val Trp Met Lys Arg Met Arg Gln Glu Arg 210 215 220 Arg Leu Cys Cys Val Val Val Leu Glu Pro Val Glu Arg Val Ser Thr 225 230 235 240 Trp Val Ala Phe Gln Ser Asp Gly Thr Val Thr Ser Cys Asp Ser Leu 245 250 255 Phe Ala His Leu His Gly Tyr Val Ser Gly Glu Asp Val Ala Gly Gln 260 265 270 His Ile Thr Asp Leu Ile Pro Ser Val Gln Leu Pro Pro Ser Gly Gln 275 280 285 His Ile Pro Lys Asn Leu Lys Ile Gln Arg Ser Val Gly Arg Ala Arg 290 295 300 Asp Gly Thr Thr Phe Pro Leu Ser Leu Lys Leu Lys Ser Gln Pro Ser 305 310 315 320 Ser Glu Glu Ala Thr Thr Gly Glu Ala Ala Pro Val Ser Gly Tyr Arg 325 330 335 Ala Ser Val Trp Val Phe Cys Thr Ile Ser Gly Leu Ile Thr Leu Leu 340 345 350 Pro Asp Gly Thr Ile His Gly Ile Asn His Ser Phe Ala Leu Thr Leu 355 360 365 Phe Gly Tyr Gly Lys Thr Glu Leu Leu Gly Lys Asn Ile Thr Phe Leu 370 375 380 Ile Pro Gly Phe Tyr Ser Tyr Met Asp Leu Ala Tyr Asn Ser Ser Leu 385 390 395 400 Gln Leu Pro Asp Leu Ala Ser Cys Leu Asp Val Gly Asn Glu Ser Gly 405 410 415 Cys Gly Glu Arg Thr Leu Asp Pro Trp Gln Gly Gln Asp Pro Ala Glu 420 425 430 Gly Gly Gln Asp Pro Arg Ile Asn Val Val Leu Ala Gly Gly His Val 435 440 445 Val Pro Arg Asp Glu Ile Arg Lys Leu Met Glu Ser Gln Asp Ile Phe 450 455 460 Thr Gly Thr Gln Thr Glu Leu Ile Ala Gly Gly Gln Leu Leu Ser Cys 465 470 475 480 Leu Ser Pro Gln Pro Ala Pro Gly Val Asp Asn Val Pro Glu Gly Ser 485 490 495 Leu Pro Val His Gly Glu Gln Ala Leu Pro Lys Asp Gln Gln Ile Thr 500 505 510 Ala Leu Gly Arg Glu Glu Pro Val Ala Ile Glu Ser Pro Gly Gln Asp 515 520 525 Leu Leu Gly Glu Ser Arg Ser Glu Pro Val Asp Val Lys Pro Phe Ala 530 535 540 Ser Cys Glu Asp Ser Glu Ala Pro Val Pro Ala Glu Asp Gly Gly Ser 545 550 555 560 Asp Ala Gly Met Cys Gly Leu Cys Gln Lys Ala Gln Leu Glu Arg Met 565 570 575 Gly Val Ser Gly Pro Ser Gly Ser Asp Leu Trp Ala Gly Ala Ala Val 580 585 590 Ala Lys Pro Gln Ala Lys Gly Gln Leu Ala Gly Gly Ser Leu Leu Met 595 600 605 His Cys Pro Cys Tyr Gly Ser Glu Trp Gly Leu Trp Trp Arg Ser Gln 610 615 620 Asp Leu Ala Pro Ser Pro Ser Gly Met Ala Gly Leu Ser Phe Gly Thr 625 630 635 640 Pro Thr Leu Asp Glu Pro Trp Leu Gly Val Glu Asn Asp Arg Glu Glu 645 650 655 Leu Gln Thr Cys Leu Ile Lys Glu Gln Leu Ser Gln Leu Ser Leu Ala 660 665 670 Gly Ala Leu Asp Val Pro His Ala Glu Leu Val Pro Thr Glu Cys Gln 675 680 685 Ala Val Thr Ala Pro Val Ser Ser Cys Asp Leu Gly Gly Arg Asp Leu 690 695 700 Cys Gly Gly Cys Thr Gly Ser Ser Ser Ala Cys Tyr Ala Leu Ala Thr 705 710 715 720 Asp Leu Pro Gly Gly Leu Glu Ala Val Glu Ala Gln Glu Val Asp Val 725 730 735 Asn Ser Phe Ser Trp Asn Leu Lys Glu Leu Phe Phe Ser Asp Gln Thr 740 745 750 Asp Gln Thr Ser Ser Asn Cys Ser Cys Ala Thr Ser Glu Leu Arg Glu 755 760 765 Thr Pro Ser Ser Leu Ala Val Gly Ser Asp Pro Asp Val Gly Ser Leu 770 775 780 Gln Glu Gln Gly Ser Cys Val Leu Asp Asp Arg Glu Leu Leu Leu Leu 785 790 795 800 Thr Gly Thr Cys Val Asp Leu Gly Gln Gly Arg Arg Phe Arg Glu Ser 805 810 815 Cys Val Gly His Asp Pro Thr Glu Pro Leu Glu Val Cys Leu Val Ser 820 825 830 Ser Glu His Tyr Ala Ala Ser Asp Arg Glu Ser Pro Gly His Val Pro 835 840 845 Ser Thr Leu Asp Ala Gly Pro Glu Asp Thr Cys Pro Ser Ala Glu Glu 850 855 860 Pro Arg Leu Asn Val Gln Val Thr Ser Thr Pro Val Ile Val Met Arg 865 870 875 880 Gly Ala Ala Gly Leu Gln Arg Glu Ile Gln Glu Gly Ala Tyr Ser Gly 885 890 895 Ser Cys His His Arg Asp Gly Leu Arg Leu Ser Ile Gln Phe Glu Val 900 905 910 Arg Arg Val Glu Leu Gln Gly Pro Thr Pro Leu Phe Cys Cys Trp Leu 915 920 925 Val Lys Asp Leu Leu His Ser Gln Arg Asp Ser Ala Ala Arg Thr Arg 930 935 940 Leu Phe Leu Ala Ser Leu Pro Gly Ser Thr His Ser Thr Ala Ala Glu 945 950 955 960 Leu Thr Gly Pro Ser Leu Val Glu Val Leu Arg Ala Arg Pro Trp Phe 965 970 975 Glu Glu Pro Pro Lys Ala Val Glu Leu Glu Gly Leu Ala Ala Cys Glu 980 985 990 Gly Glu Tyr Ser Gln Lys Tyr Ser Thr Met Ser Pro Leu Gly Ser Gly 995 1000 1005 Ala Phe Gly Phe Val Trp Thr Ala Val Asp Lys Glu Lys Asn Lys 1010 1015 1020 Glu Val Val Val Lys Phe Ile Lys Lys Glu Lys Val Leu Glu Asp 1025 1030 1035 Cys Trp Ile Glu Asp Pro Lys Leu Gly Lys Val Thr Leu Glu Ile 1040 1045 1050 Ala Ile Leu Ser Arg Val Glu His Ala Asn Ile Ile Lys Val Leu 1055 1060 1065 Asp Ile Phe Glu Asn Gln Gly Phe Phe Gln Leu Val Met Glu Lys 1070 1075 1080 His Gly Ser Gly Leu Asp Leu Phe Ala Phe Ile Asp Arg His Pro 1085 1090 1095 Arg Leu Asp Glu Pro Leu Ala Ser Tyr Ile Phe Arg Gln Leu Val 1100 1105 1110 Ser Ala Val Gly Tyr Leu Arg Leu Lys Asp Ile Ile His Arg Asp 1115 1120 1125 Ile Lys Asp Glu Asn Ile Val Ile Ala Glu Asp Phe Thr Ile Lys 1130 1135 1140 Leu Ile Asp Phe Gly Ser Ala Ala Tyr Leu Glu Arg Gly Lys Leu 1145 1150 1155 Phe Tyr Thr Phe Cys Gly Thr Ile Glu Tyr Cys Ala Pro Glu Val 1160 1165 1170 Leu Met Gly Asn Pro Tyr Arg Gly Pro Glu Leu Glu Met Trp Ser 1175 1180 1185 Leu Gly Val Thr Leu Tyr Thr Leu Val Phe Glu Glu Asn Pro Phe 1190 1195 1200 Cys Glu Leu Glu Glu Thr Val Glu Ala Ala Ile His Pro Pro Tyr 1205 1210 1215 Leu Val Ser Lys Glu Leu Met Ser Leu Val Ser Gly Leu Leu Gln 1220 1225 1230 Pro Val Pro Glu Arg Arg Thr Thr Leu Glu Lys Leu Val Thr Asp 1235 1240 1245 Pro Trp Val Thr Gln Pro Val Asn Leu Ala Asp Tyr Thr Trp Glu 1250 1255 1260 Glu Val Cys Arg Val Asn Lys Pro Glu Ser Gly Val Leu Ser Ala 1265 1270 1275 Ala Ser Leu Glu Met Gly Asn Arg Ser Leu Ser Asp Val Ala Gln 1280 1285 1290 Ala Gln Glu Leu Cys Gly Gly Pro Val Pro Gly Glu Ala Pro Asn 1295 1300 1305 Gly Gln Gly Cys Leu His Pro Gly Asp Pro Arg Leu Leu Thr Ser 1310 1315 1320 3 3271 DNA Drosophila melanogaster CDS (121)..(3135) 3 atgcgccatt caattgattt tctacgagca gagttcccaa tgtttacggt gcgctcattt 60 ggttgtagct gtaggcctcg agttggaacc gctgggactt ggaaaacata cgacgcttaa 120 atg gag gat cag ggc gac gtg aca gac caa gcc gct ggt aag ctg agt 168 Met Glu Asp Gln Gly Asp Val Thr Asp Gln Ala Ala Gly Lys Leu Ser 1 5 10 15 agt tcg aat gcc cag tgc tcc aga aac cga tgt gct aat gat gac ccc 216 Ser Ser Asn Ala Gln Cys Ser Arg Asn Arg Cys Ala Asn Asp Asp Pro 20 25 30 att atg ggc agc cac agt cag agt cgc cag aac tcg tcc ggc atg ctg 264 Ile Met Gly Ser His Ser Gln Ser Arg Gln Asn Ser Ser Gly Met Leu 35 40 45 aac atg atg gac gcc agc tcc ttc agc atg ccc ccc ccc aat ctg cac 312 Asn Met Met Asp Ala Ser Ser Phe Ser Met Pro Pro Pro Asn Leu His 50 55 60 tcc tca aag gtg atc aac ccg aac aag gcg ata ttc aca atc gac gcg 360 Ser Ser Lys Val Ile Asn Pro Asn Lys Ala Ile Phe Thr Ile Asp Ala 65 70 75 80 aac aca ggg caa att ttt ata gtg aac aac aag gcc tgc cag ctg ctg 408 Asn Thr Gly Gln Ile Phe Ile Val Asn Asn Lys Ala Cys Gln Leu Leu 85 90 95 ggc tac acg tcg cag gag ttg agg aac aag gga ttc ttc gac ctg ctt 456 Gly Tyr Thr Ser Gln Glu Leu Arg Asn Lys Gly Phe Phe Asp Leu Leu 100 105 110 aac ggc aag aca gag agc cac atc tcc tcc ctg gcg gag atg cag atc 504 Asn Gly Lys Thr Glu Ser His Ile Ser Ser Leu Ala Glu Met Gln Ile 115 120 125 gaa ggc gat gag ggc cgg gtc gtc ctg cta agt gga aag gtc atc gag 552 Glu Gly Asp Glu Gly Arg Val Val Leu Leu Ser Gly Lys Val Ile Glu 130 135 140 atg aag acc aag tcg ggc ggc aag atc ctg gtc tct ctc tgg atc cgg 600 Met Lys Thr Lys Ser Gly Gly Lys Ile Leu Val Ser Leu Trp Ile Arg 145 150 155 160 cag atc agc agc gat ggc cga cac ata gcc gtc gcg gag cct gtg gag 648 Gln Ile Ser Ser Asp Gly Arg His Ile Ala Val Ala Glu Pro Val Glu 165 170 175 agg cat att tgc cac atc agc acc gat aga tcc gga gtg ata aca tcc 696 Arg His Ile Cys His Ile Ser Thr Asp Arg Ser Gly Val Ile Thr Ser 180 185 190 gtt gac tcc acc acg gcc acc ata ttc ttt tat gaa tcc gtc gaa agc 744 Val Asp Ser Thr Thr Ala Thr Ile Phe Phe Tyr Glu Ser Val Glu Ser 195 200 205 gtg gtg ggt gtc agc att gtc acc ctt atc ccg ttt ata aag ctg ccc 792 Val Val Gly Val Ser Ile Val Thr Leu Ile Pro Phe Ile Lys Leu Pro 210 215 220 gat ccg gat agc cgt gag atc ccc aag agc ctg cgg aag cag aga gct 840 Asp Pro Asp Ser Arg Glu Ile Pro Lys Ser Leu Arg Lys Gln Arg Ala 225 230 235 240 act ggt cga act acg gat aac gta aag ttt ccc ctg tgc ttg cta ata 888 Thr Gly Arg Thr Thr Asp Asn Val Lys Phe Pro Leu Cys Leu Leu Ile 245 250 255 gcc ctg gac gag gat gct tct gcg ggg tat tcc cat tcg gga aaa acc 936 Ala Leu Asp Glu Asp Ala Ser Ala Gly Tyr Ser His Ser Gly Lys Thr 260 265 270 ggt cta aat atc acc gtc tgg gtg ttc cag aat ctt agt ggc ctg att 984 Gly Leu Asn Ile Thr Val Trp Val Phe Gln Asn Leu Ser Gly Leu Ile 275 280 285 gtg gta gat gac ata ggc aac ata ctg atg tgc aac cag cct ttc tcg 1032 Val Val Asp Asp Ile Gly Asn Ile Leu Met Cys Asn Gln Pro Phe Ser 290 295 300 ctg cta atg ttc gga tat ggt cag gac aag ata atg aac atg cat ata 1080 Leu Leu Met Phe Gly Tyr Gly Gln Asp Lys Ile Met Asn Met His Ile 305 310 315 320 tcg gcg atc cta ccc aat ttc ggc aag gac tct cgc gag gag aag agt 1128 Ser Ala Ile Leu Pro Asn Phe Gly Lys Asp Ser Arg Glu Glu Lys Ser 325 330 335 ccg aat gtt tcg aac acc tcg ata acc agt aac gac tgg gag ccg gac 1176 Pro Asn Val Ser Asn Thr Ser Ile Thr Ser Asn Asp Trp Glu Pro Asp 340 345 350 acg gat ccc ctc gtc gtg gac aac gac tcc tcg ctg caa tcc tgc aag 1224 Thr Asp Pro Leu Val Val Asp Asn Asp Ser Ser Leu Gln Ser Cys Lys 355 360 365 aag agc tcg act aac agg acg gcg ccc aac aac gaa cag tct tcg gtg 1272 Lys Ser Ser Thr Asn Arg Thr Ala Pro Asn Asn Glu Gln Ser Ser Val 370 375 380 ggc gat gcc cac cta agc tgc aat ctg gag aac agc agt ggt cta ttc 1320 Gly Asp Ala His Leu Ser Cys Asn Leu Glu Asn Ser Ser Gly Leu Phe 385 390 395 400 tgc gac ctc cgc cag cca gat gat tgt aca ata gat gac att cta aca 1368 Cys Asp Leu Arg Gln Pro Asp Asp Cys Thr Ile Asp Asp Ile Leu Thr 405 410 415 cct gta aat gcc tca aac agt ttc ccc gct gac gag ttt gag gta gga 1416 Pro Val Asn Ala Ser Asn Ser Phe Pro Ala Asp Glu Phe Glu Val Gly 420 425 430 tcg cag tcg cag gtg aat gag tcg tcc ctt gac aag gcg aag tcc gcg 1464 Ser Gln Ser Gln Val Asn Glu Ser Ser Leu Asp Lys Ala Lys Ser Ala 435 440 445 tcc acg gag acc tgt gat gcc tcc ggc agc aat ccc gcc acc aga ctg 1512 Ser Thr Glu Thr Cys Asp Ala Ser Gly Ser Asn Pro Ala Thr Arg Leu 450 455 460 ctt agt tcc atc aac gga agc ttt gtg ggg gag gcc att cac gcc gat 1560 Leu Ser Ser Ile Asn Gly Ser Phe Val Gly Glu Ala Ile His Ala Asp 465 470 475 480 ggc tcc gtc ata gag gta gtc tac tcc gtg ctc ctc cag ata ctg ccc 1608 Gly Ser Val Ile Glu Val Val Tyr Ser Val Leu Leu Gln Ile Leu Pro 485 490 495 tgc tcc aac cgg gtg tac tgc atc tgg gtg tgc cgc aac ccc agc act 1656 Cys Ser Asn Arg Val Tyr Cys Ile Trp Val Cys Arg Asn Pro Ser Thr 500 505 510 cgg ctg gac ggt gag aag tac aac tat gca aac cta acg tcc aca ttc 1704 Arg Leu Asp Gly Glu Lys Tyr Asn Tyr Ala Asn Leu Thr Ser Thr Phe 515 520 525 aac agc atg gcc agc act gtt gag cag tcg ctg ggc cag gtg ata aag 1752 Asn Ser Met Ala Ser Thr Val Glu Gln Ser Leu Gly Gln Val Ile Lys 530 535 540 acc acc gct gcc cag aac tcc agc aga ccc aat tct ctg tcc ttg gtg 1800 Thr Thr Ala Ala Gln Asn Ser Ser Arg Pro Asn Ser Leu Ser Leu Val 545 550 555 560 tcc aag tac gag gac gaa ttg tac ctg ggc gac tac agc aaa tac tac 1848 Ser Lys Tyr Glu Asp Glu Leu Tyr Leu Gly Asp Tyr Ser Lys Tyr Tyr 565 570 575 acg tcc atc cgc cag atc gga aga gga gcc tat ggg tat gtg aat atg 1896 Thr Ser Ile Arg Gln Ile Gly Arg Gly Ala Tyr Gly Tyr Val Asn Met 580 585 590 gct ttc cgg aac agc gac cgc ctg ctg gtg atc acc aag ttt att ctc 1944 Ala Phe Arg Asn Ser Asp Arg Leu Leu Val Ile Thr Lys Phe Ile Leu 595 600 605 aag gag aag ctc tgc tct cag ttt atg gtt aag agc cgc gac tgc aag 1992 Lys Glu Lys Leu Cys Ser Gln Phe Met Val Lys Ser Arg Asp Cys Lys 610 615 620 gaa gtt cct atc gag atc cac ctg ctg cag acc ctg aac cac aag aac 2040 Glu Val Pro Ile Glu Ile His Leu Leu Gln Thr Leu Asn His Lys Asn 625 630 635 640 att gtg tcc gtt ctc gat gtt ttc gaa aat gat cta ttc tac cag ctg 2088 Ile Val Ser Val Leu Asp Val Phe Glu Asn Asp Leu Phe Tyr Gln Leu 645 650 655 gtc atg gag aag cac gga tcc gga atg gat cta tgg acg ttc ata gag 2136 Val Met Glu Lys His Gly Ser Gly Met Asp Leu Trp Thr Phe Ile Glu 660 665 670 cgg cgg ccc ctg atg gac gaa aag ctg gga agc tac att ttt cgt cag 2184 Arg Arg Pro Leu Met Asp Glu Lys Leu Gly Ser Tyr Ile Phe Arg Gln 675 680 685 gtg gcc gat gcc gtc aac tat ctg cac gaa cag aag ata ctg cat cgg 2232 Val Ala Asp Ala Val Asn Tyr Leu His Glu Gln Lys Ile Leu His Arg 690 695 700 gac atc aag gac gag aac att ata att gac cag aac ttc acc ata aag 2280 Asp Ile Lys Asp Glu Asn Ile Ile Ile Asp Gln Asn Phe Thr Ile Lys 705 710 715 720 ctg att gac ttc gga tcg gcg acc ttc atg gag gag gga aaa ttc ttc 2328 Leu Ile Asp Phe Gly Ser Ala Thr Phe Met Glu Glu Gly Lys Phe Phe 725 730 735 tcc acg ttc tac ggc acg acg gag tac tgc agt ccg gag gtt ctg gcc 2376 Ser Thr Phe Tyr Gly Thr Thr Glu Tyr Cys Ser Pro Glu Val Leu Ala 740 745 750 gga aac aga tac gtg ggt ccc gag ctg gag att tgg gcc ctt ggt gtg 2424 Gly Asn Arg Tyr Val Gly Pro Glu Leu Glu Ile Trp Ala Leu Gly Val 755 760 765 acg ctt tac gtt ctg atg ttt ttc gaa aat cca ttc atc gat gtg gag 2472 Thr Leu Tyr Val Leu Met Phe Phe Glu Asn Pro Phe Ile Asp Val Glu 770 775 780 gag aca ttg aag gcc gag att caa ata ccg aaa gct gtg tcc gag caa 2520 Glu Thr Leu Lys Ala Glu Ile Gln Ile Pro Lys Ala Val Ser Glu Gln 785 790 795 800 ttg agc cgt ctg ttg agc tcc atg ctg aat aag gat ccc aag tac cgg 2568 Leu Ser Arg Leu Leu Ser Ser Met Leu Asn Lys Asp Pro Lys Tyr Arg 805 810 815 tgc acc atg cac cag tta atc aca gat cca tgg ctc acc cag gag gtg 2616 Cys Thr Met His Gln Leu Ile Thr Asp Pro Trp Leu Thr Gln Glu Val 820 825 830 aat ccc tct act ttc agt ttc tcg tgg ata gtg ccg tgc aag gcc cac 2664 Asn Pro Ser Thr Phe Ser Phe Ser Trp Ile Val Pro Cys Lys Ala His 835 840 845 gag gcc aat ccg aat ctg tat ttt tcc ggc tac cta tac tcc agt act 2712 Glu Ala Asn Pro Asn Leu Tyr Phe Ser Gly Tyr Leu Tyr Ser Ser Thr 850 855 860 tcg gtg ctg tcc act att tca ccg cag gag agc ttc tcc cac atc gag 2760 Ser Val Leu Ser Thr Ile Ser Pro Gln Glu Ser Phe Ser His Ile Glu 865 870 875 880 gag tcc tcg att ggg ggc agc gat gac gcc aga tta gca tcc cac aga 2808 Glu Ser Ser Ile Gly Gly Ser Asp Asp Ala Arg Leu Ala Ser His Arg 885 890 895 acc gga cac aag ttg tgc gtt aat gaa ggt aca ata ctt ctt cgt ctg 2856 Thr Gly His Lys Leu Cys Val Asn Glu Gly Thr Ile Leu Leu Arg Leu 900 905 910 ctg cgt att tta gtt gta aag cat ccc ttt gtc att cta gcc aag cac 2904 Leu Arg Ile Leu Val Val Lys His Pro Phe Val Ile Leu Ala Lys His 915 920 925 aac aaa atg gac act ctt tac gcg aaa cag ttc cag cta aac acc tca 2952 Asn Lys Met Asp Thr Leu Tyr Ala Lys Gln Phe Gln Leu Asn Thr Ser 930 935 940 gtg agt aac cac gag ctg agg gtc tcg tta cct gac tca agc cac aca 3000 Val Ser Asn His Glu Leu Arg Val Ser Leu Pro Asp Ser Ser His Thr 945 950 955 960 gaa atc ggc ggt tca ata tgc tcc tcg aaa tcc gag aac gat ata ttc 3048 Glu Ile Gly Gly Ser Ile Cys Ser Ser Lys Ser Glu Asn Asp Ile Phe 965 970 975 aag aac aag ctt cag ccc tgc gtc tcc act tat aac gta gtt agt ctg 3096 Lys Asn Lys Leu Gln Pro Cys Val Ser Thr Tyr Asn Val Val Ser Leu 980 985 990 cac gac gtg agc acg aaa ccc aag aag ctt gat ctg aaa tgaagttcga 3145 His Asp Val Ser Thr Lys Pro Lys Lys Leu Asp Leu Lys 995 1000 1005 aatccgttgt gtacatgtgt aaatcgaatc tgagcatact gtcatatgta aatagcaaca 3205 tttgtgaaag gacaattaag tattggggcg gaaaagcatg tagatgaata aatcaattta 3265 attgtt 3271 4 1005 PRT Drosophila melanogaster 4 Met Glu Asp Gln Gly Asp Val Thr Asp Gln Ala Ala Gly Lys Leu Ser 1 5 10 15 Ser Ser Asn Ala Gln Cys Ser Arg Asn Arg Cys Ala Asn Asp Asp Pro 20 25 30 Ile Met Gly Ser His Ser Gln Ser Arg Gln Asn Ser Ser Gly Met Leu 35 40 45 Asn Met Met Asp Ala Ser Ser Phe Ser Met Pro Pro Pro Asn Leu His 50 55 60 Ser Ser Lys Val Ile Asn Pro Asn Lys Ala Ile Phe Thr Ile Asp Ala 65 70 75 80 Asn Thr Gly Gln Ile Phe Ile Val Asn Asn Lys Ala Cys Gln Leu Leu 85 90 95 Gly Tyr Thr Ser Gln Glu Leu Arg Asn Lys Gly Phe Phe Asp Leu Leu 100 105 110 Asn Gly Lys Thr Glu Ser His Ile Ser Ser Leu Ala Glu Met Gln Ile 115 120 125 Glu Gly Asp Glu Gly Arg Val Val Leu Leu Ser Gly Lys Val Ile Glu 130 135 140 Met Lys Thr Lys Ser Gly Gly Lys Ile Leu Val Ser Leu Trp Ile Arg 145 150 155 160 Gln Ile Ser Ser Asp Gly Arg His Ile Ala Val Ala Glu Pro Val Glu 165 170 175 Arg His Ile Cys His Ile Ser Thr Asp Arg Ser Gly Val Ile Thr Ser 180 185 190 Val Asp Ser Thr Thr Ala Thr Ile Phe Phe Tyr Glu Ser Val Glu Ser 195 200 205 Val Val Gly Val Ser Ile Val Thr Leu Ile Pro Phe Ile Lys Leu Pro 210 215 220 Asp Pro Asp Ser Arg Glu Ile Pro Lys Ser Leu Arg Lys Gln Arg Ala 225 230 235 240 Thr Gly Arg Thr Thr Asp Asn Val Lys Phe Pro Leu Cys Leu Leu Ile 245 250 255 Ala Leu Asp Glu Asp Ala Ser Ala Gly Tyr Ser His Ser Gly Lys Thr 260 265 270 Gly Leu Asn Ile Thr Val Trp Val Phe Gln Asn Leu Ser Gly Leu Ile 275 280 285 Val Val Asp Asp Ile Gly Asn Ile Leu Met Cys Asn Gln Pro Phe Ser 290 295 300 Leu Leu Met Phe Gly Tyr Gly Gln Asp Lys Ile Met Asn Met His Ile 305 310 315 320 Ser Ala Ile Leu Pro Asn Phe Gly Lys Asp Ser Arg Glu Glu Lys Ser 325 330 335 Pro Asn Val Ser Asn Thr Ser Ile Thr Ser Asn Asp Trp Glu Pro Asp 340 345 350 Thr Asp Pro Leu Val Val Asp Asn Asp Ser Ser Leu Gln Ser Cys Lys 355 360 365 Lys Ser Ser Thr Asn Arg Thr Ala Pro Asn Asn Glu Gln Ser Ser Val 370 375 380 Gly Asp Ala His Leu Ser Cys Asn Leu Glu Asn Ser Ser Gly Leu Phe 385 390 395 400 Cys Asp Leu Arg Gln Pro Asp Asp Cys Thr Ile Asp Asp Ile Leu Thr 405 410 415 Pro Val Asn Ala Ser Asn Ser Phe Pro Ala Asp Glu Phe Glu Val Gly 420 425 430 Ser Gln Ser Gln Val Asn Glu Ser Ser Leu Asp Lys Ala Lys Ser Ala 435 440 445 Ser Thr Glu Thr Cys Asp Ala Ser Gly Ser Asn Pro Ala Thr Arg Leu 450 455 460 Leu Ser Ser Ile Asn Gly Ser Phe Val Gly Glu Ala Ile His Ala Asp 465 470 475 480 Gly Ser Val Ile Glu Val Val Tyr Ser Val Leu Leu Gln Ile Leu Pro 485 490 495 Cys Ser Asn Arg Val Tyr Cys Ile Trp Val Cys Arg Asn Pro Ser Thr 500 505 510 Arg Leu Asp Gly Glu Lys Tyr Asn Tyr Ala Asn Leu Thr Ser Thr Phe 515 520 525 Asn Ser Met Ala Ser Thr Val Glu Gln Ser Leu Gly Gln Val Ile Lys 530 535 540 Thr Thr Ala Ala Gln Asn Ser Ser Arg Pro Asn Ser Leu Ser Leu Val 545 550 555 560 Ser Lys Tyr Glu Asp Glu Leu Tyr Leu Gly Asp Tyr Ser Lys Tyr Tyr 565 570 575 Thr Ser Ile Arg Gln Ile Gly Arg Gly Ala Tyr Gly Tyr Val Asn Met 580 585 590 Ala Phe Arg Asn Ser Asp Arg Leu Leu Val Ile Thr Lys Phe Ile Leu 595 600 605 Lys Glu Lys Leu Cys Ser Gln Phe Met Val Lys Ser Arg Asp Cys Lys 610 615 620 Glu Val Pro Ile Glu Ile His Leu Leu Gln Thr Leu Asn His Lys Asn 625 630 635 640 Ile Val Ser Val Leu Asp Val Phe Glu Asn Asp Leu Phe Tyr Gln Leu 645 650 655 Val Met Glu Lys His Gly Ser Gly Met Asp Leu Trp Thr Phe Ile Glu 660 665 670 Arg Arg Pro Leu Met Asp Glu Lys Leu Gly Ser Tyr Ile Phe Arg Gln 675 680 685 Val Ala Asp Ala Val Asn Tyr Leu His Glu Gln Lys Ile Leu His Arg 690 695 700 Asp Ile Lys Asp Glu Asn Ile Ile Ile Asp Gln Asn Phe Thr Ile Lys 705 710 715 720 Leu Ile Asp Phe Gly Ser Ala Thr Phe Met Glu Glu Gly Lys Phe Phe 725 730 735 Ser Thr Phe Tyr Gly Thr Thr Glu Tyr Cys Ser Pro Glu Val Leu Ala 740 745 750 Gly Asn Arg Tyr Val Gly Pro Glu Leu Glu Ile Trp Ala Leu Gly Val 755 760 765 Thr Leu Tyr Val Leu Met Phe Phe Glu Asn Pro Phe Ile Asp Val Glu 770 775 780 Glu Thr Leu Lys Ala Glu Ile Gln Ile Pro Lys Ala Val Ser Glu Gln 785 790 795 800 Leu Ser Arg Leu Leu Ser Ser Met Leu Asn Lys Asp Pro Lys Tyr Arg 805 810 815 Cys Thr Met His Gln Leu Ile Thr Asp Pro Trp Leu Thr Gln Glu Val 820 825 830 Asn Pro Ser Thr Phe Ser Phe Ser Trp Ile Val Pro Cys Lys Ala His 835 840 845 Glu Ala Asn Pro Asn Leu Tyr Phe Ser Gly Tyr Leu Tyr Ser Ser Thr 850 855 860 Ser Val Leu Ser Thr Ile Ser Pro Gln Glu Ser Phe Ser His Ile Glu 865 870 875 880 Glu Ser Ser Ile Gly Gly Ser Asp Asp Ala Arg Leu Ala Ser His Arg 885 890 895 Thr Gly His Lys Leu Cys Val Asn Glu Gly Thr Ile Leu Leu Arg Leu 900 905 910 Leu Arg Ile Leu Val Val Lys His Pro Phe Val Ile Leu Ala Lys His 915 920 925 Asn Lys Met Asp Thr Leu Tyr Ala Lys Gln Phe Gln Leu Asn Thr Ser 930 935 940 Val Ser Asn His Glu Leu Arg Val Ser Leu Pro Asp Ser Ser His Thr 945 950 955 960 Glu Ile Gly Gly Ser Ile Cys Ser Ser Lys Ser Glu Asn Asp Ile Phe 965 970 975 Lys Asn Lys Leu Gln Pro Cys Val Ser Thr Tyr Asn Val Val Ser Leu 980 985 990 His Asp Val Ser Thr Lys Pro Lys Lys Leu Asp Leu Lys 995 1000 1005 5 4071 DNA yeast CDS (1)..(4068) 5 atg ccc tac atc ggt gct tcc aac ctc tca gaa cat tca ttt gtt aat 48 Met Pro Tyr Ile Gly Ala Ser Asn Leu Ser Glu His Ser Phe Val Asn 1 5 10 15 ttg aag gaa aaa cat gcg att aca cat aaa ggt acg agc agt tct gta 96 Leu Lys Glu Lys His Ala Ile Thr His Lys Gly Thr Ser Ser Ser Val 20 25 30 gca tct ttg cag aca cca ccg agc ccc gat caa gag aac cat att gac 144 Ala Ser Leu Gln Thr Pro Pro Ser Pro Asp Gln Glu Asn His Ile Asp 35 40 45 aat gaa tta gaa aat tac gat acg tct tta agt gac gtt tca acc ccg 192 Asn Glu Leu Glu Asn Tyr Asp Thr Ser Leu Ser Asp Val Ser Thr Pro 50 55 60 aat aaa aag gaa ggt gat gag ttc cag caa agt tta aga gat aca ttt 240 Asn Lys Lys Glu Gly Asp Glu Phe Gln Gln Ser Leu Arg Asp Thr Phe 65 70 75 80 gcg agc ttt cgg aag act aaa ccc cca cct cct tta gat ttt gaa caa 288 Ala Ser Phe Arg Lys Thr Lys Pro Pro Pro Pro Leu Asp Phe Glu Gln 85 90 95 cca aga ctt cct tcg aca gct tct tca tcc gtt gat tca acc gta tca 336 Pro Arg Leu Pro Ser Thr Ala Ser Ser Ser Val Asp Ser Thr Val Ser 100 105 110 tcg ccc tta acg gat gaa gac ata aag gag tta gag ttt ctt ccg aat 384 Ser Pro Leu Thr Asp Glu Asp Ile Lys Glu Leu Glu Phe Leu Pro Asn 115 120 125 gaa tca act cat tct tat tcg tac aat cca ctt tcg cca aat tcc ctg 432 Glu Ser Thr His Ser Tyr Ser Tyr Asn Pro Leu Ser Pro Asn Ser Leu 130 135 140 gca gtc aga ttg agg att ttg aag aga tca ttg gaa atc ata ata caa 480 Ala Val Arg Leu Arg Ile Leu Lys Arg Ser Leu Glu Ile Ile Ile Gln 145 150 155 160 aac cca agt atg cta ctg gag cct act cca gat gat ttg cct cct ttg 528 Asn Pro Ser Met Leu Leu Glu Pro Thr Pro Asp Asp Leu Pro Pro Leu 165 170 175 aaa gag ttt gcg ggc cgt agg agc agt tta cca agg aca tcg gct tct 576 Lys Glu Phe Ala Gly Arg Arg Ser Ser Leu Pro Arg Thr Ser Ala Ser 180 185 190 gca aac cat tta atg aac aga aat aag agc cag att tgg aac act act 624 Ala Asn His Leu Met Asn Arg Asn Lys Ser Gln Ile Trp Asn Thr Thr 195 200 205 tcc gct act tta aat gca ttt gta aat aat acc tct tcc tcc tca gca 672 Ser Ala Thr Leu Asn Ala Phe Val Asn Asn Thr Ser Ser Ser Ser Ala 210 215 220 gca tct tct gct tta tct aac aaa aaa ccg ggc acc cca gtt ttc cct 720 Ala Ser Ser Ala Leu Ser Asn Lys Lys Pro Gly Thr Pro Val Phe Pro 225 230 235 240 aat ttg gat cca aca cat tct caa aca ttc cat aga gcc aac tcg ttg 768 Asn Leu Asp Pro Thr His Ser Gln Thr Phe His Arg Ala Asn Ser Leu 245 250 255 gct tat tta cct tcc atc tta cct gag caa gat ccg ctg ctc aaa cat 816 Ala Tyr Leu Pro Ser Ile Leu Pro Glu Gln Asp Pro Leu Leu Lys His 260 265 270 aat aat tct tta ttt cgt ggc gac tat gga aac aac ata agt cct gaa 864 Asn Asn Ser Leu Phe Arg Gly Asp Tyr Gly Asn Asn Ile Ser Pro Glu 275 280 285 agg cca agt ttt aga caa ccc ttc aag gat caa act agc aat ctc cgc 912 Arg Pro Ser Phe Arg Gln Pro Phe Lys Asp Gln Thr Ser Asn Leu Arg 290 295 300 aat agc agt tta ctc aat gag agg gca tat cag gaa gat gaa act ttt 960 Asn Ser Ser Leu Leu Asn Glu Arg Ala Tyr Gln Glu Asp Glu Thr Phe 305 310 315 320 tta ccg cac cat gga ccc tct atg gat cta ttg aat gaa caa aga gcg 1008 Leu Pro His His Gly Pro Ser Met Asp Leu Leu Asn Glu Gln Arg Ala 325 330 335 aac ttg aaa agt ctt ctg aat tta tta aac gaa aca ctg gag aaa aat 1056 Asn Leu Lys Ser Leu Leu Asn Leu Leu Asn Glu Thr Leu Glu Lys Asn 340 345 350 act tcc gag aga gct tcg gat ctt cat atg ata tcg ttg ttc aat ttg 1104 Thr Ser Glu Arg Ala Ser Asp Leu His Met Ile Ser Leu Phe Asn Leu 355 360 365 aat aaa cta atg ctt gga gat ccc aag aaa aat aat tca gaa cgc gat 1152 Asn Lys Leu Met Leu Gly Asp Pro Lys Lys Asn Asn Ser Glu Arg Asp 370 375 380 aaa aga act gaa aag ctt aaa aag att ttg ctg gat agt ctt gca gaa 1200 Lys Arg Thr Glu Lys Leu Lys Lys Ile Leu Leu Asp Ser Leu Ala Glu 385 390 395 400 cca ttc ttt gag cac tat aat ttt att gga gat aat ccg atc gca gat 1248 Pro Phe Phe Glu His Tyr Asn Phe Ile Gly Asp Asn Pro Ile Ala Asp 405 410 415 aca gat gaa cta aag gag gaa att gat gaa ttt aca ggt tct gga gat 1296 Thr Asp Glu Leu Lys Glu Glu Ile Asp Glu Phe Thr Gly Ser Gly Asp 420 425 430 acg aca gcg ata aca gat ata cgg ccc caa cag gac tat ggc cgc ata 1344 Thr Thr Ala Ile Thr Asp Ile Arg Pro Gln Gln Asp Tyr Gly Arg Ile 435 440 445 ttg agg aca ttc act tct acc aaa aat tcc gcc cca cag gca att ttt 1392 Leu Arg Thr Phe Thr Ser Thr Lys Asn Ser Ala Pro Gln Ala Ile Phe 450 455 460 aca tgt agt cag gaa gac cct tgg caa ttc aga gct gcg aat gat cta 1440 Thr Cys Ser Gln Glu Asp Pro Trp Gln Phe Arg Ala Ala Asn Asp Leu 465 470 475 480 gcg tgc tta gta ttc ggt atc tca cag aat gcc att cgc gct tta acc 1488 Ala Cys Leu Val Phe Gly Ile Ser Gln Asn Ala Ile Arg Ala Leu Thr 485 490 495 ttg atg gat tta att cac acc gat agc aga aat ttt gtt tta cac aaa 1536 Leu Met Asp Leu Ile His Thr Asp Ser Arg Asn Phe Val Leu His Lys 500 505 510 tta ctt tct acg gag ggt caa gaa atg gtt ttc aca ggc gaa atc att 1584 Leu Leu Ser Thr Glu Gly Gln Glu Met Val Phe Thr Gly Glu Ile Ile 515 520 525 ggt ata gtt caa cca gaa aca ctc agc tca tcc aaa gta gta tgg gca 1632 Gly Ile Val Gln Pro Glu Thr Leu Ser Ser Ser Lys Val Val Trp Ala 530 535 540 tcg ttt tgg gca aaa agg aaa aac ggc tta tta gtt tgt gtt ttc gaa 1680 Ser Phe Trp Ala Lys Arg Lys Asn Gly Leu Leu Val Cys Val Phe Glu 545 550 555 560 aag gtt cct tgc gat tat gtt gat gta ctt ttg aac ctg gat gat ttt 1728 Lys Val Pro Cys Asp Tyr Val Asp Val Leu Leu Asn Leu Asp Asp Phe 565 570 575 ggg gcc gag aat att gta gac aaa tgt gag tta tta tca gat gga ccc 1776 Gly Ala Glu Asn Ile Val Asp Lys Cys Glu Leu Leu Ser Asp Gly Pro 580 585 590 aca ttg tct tcc tct tct aca tta tcg cta cct aag atg gct tct tca 1824 Thr Leu Ser Ser Ser Ser Thr Leu Ser Leu Pro Lys Met Ala Ser Ser 595 600 605 cca act ggt agt aaa tta gag tat tcg ttg gag agg aaa atc ctg gaa 1872 Pro Thr Gly Ser Lys Leu Glu Tyr Ser Leu Glu Arg Lys Ile Leu Glu 610 615 620 aag agt tat act aag cct act tca aca gag aat cgc aac ggc gat gaa 1920 Lys Ser Tyr Thr Lys Pro Thr Ser Thr Glu Asn Arg Asn Gly Asp Glu 625 630 635 640 aac caa ctt gat gga gat agt cat tct gaa cca tcg ctg tca tca tcg 1968 Asn Gln Leu Asp Gly Asp Ser His Ser Glu Pro Ser Leu Ser Ser Ser 645 650 655 cca gta agg tcg aag aaa agt gta aag ttc gca aat gat att aaa gac 2016 Pro Val Arg Ser Lys Lys Ser Val Lys Phe Ala Asn Asp Ile Lys Asp 660 665 670 gtc aag agt ata agc caa tcg tta gcc aaa tta atg gat gat gtg agg 2064 Val Lys Ser Ile Ser Gln Ser Leu Ala Lys Leu Met Asp Asp Val Arg 675 680 685 aat ggg gtt gta ttt gat ccc gat gac gac ctt ttg cct atg ccc atc 2112 Asn Gly Val Val Phe Asp Pro Asp Asp Asp Leu Leu Pro Met Pro Ile 690 695 700 aaa gtt tgc aac cac att aat gaa aca aga tat ttt act cta aat cat 2160 Lys Val Cys Asn His Ile Asn Glu Thr Arg Tyr Phe Thr Leu Asn His 705 710 715 720 cta tct tat aat atc cca tgc gcg gtt tcc tcc act gtg ttg gag gat 2208 Leu Ser Tyr Asn Ile Pro Cys Ala Val Ser Ser Thr Val Leu Glu Asp 725 730 735 gag ctg aaa tta aag att cac agt tta cct tac cag gcg ggt ttg ttt 2256 Glu Leu Lys Leu Lys Ile His Ser Leu Pro Tyr Gln Ala Gly Leu Phe 740 745 750 att gtg gat tcg cat act tta gat att gta agt tcc aat aaa tct att 2304 Ile Val Asp Ser His Thr Leu Asp Ile Val Ser Ser Asn Lys Ser Ile 755 760 765 tta aaa aac atg ttt ggt tat cat ttt gct gag ctg gtg gga aaa tcc 2352 Leu Lys Asn Met Phe Gly Tyr His Phe Ala Glu Leu Val Gly Lys Ser 770 775 780 att act gaa ata att cct tct ttc cca aaa ttc ctc caa ttt ata aat 2400 Ile Thr Glu Ile Ile Pro Ser Phe Pro Lys Phe Leu Gln Phe Ile Asn 785 790 795 800 gac aaa tat cct gcg ttg gat atc aca ctc cat aaa aat aaa ggt ttg 2448 Asp Lys Tyr Pro Ala Leu Asp Ile Thr Leu His Lys Asn Lys Gly Leu 805 810 815 gta tta aca gaa cat ttt ttt agg aaa att cag gca gag att atg ggt 2496 Val Leu Thr Glu His Phe Phe Arg Lys Ile Gln Ala Glu Ile Met Gly 820 825 830 gat cgt aaa agc ttt tat acg tcg gtg ggt att gat ggc ctt cat agg 2544 Asp Arg Lys Ser Phe Tyr Thr Ser Val Gly Ile Asp Gly Leu His Arg 835 840 845 gat ggc tgt gaa atc aaa att gat ttc cag ctg cgt gtc atg aat tct 2592 Asp Gly Cys Glu Ile Lys Ile Asp Phe Gln Leu Arg Val Met Asn Ser 850 855 860 aaa gtg att ttg ctt tgg gtt aca cat tcg aga gac gtg gta ttt gaa 2640 Lys Val Ile Leu Leu Trp Val Thr His Ser Arg Asp Val Val Phe Glu 865 870 875 880 gaa tat aat aca aat cca tct caa ttg aag atg ctg aag gag agt gaa 2688 Glu Tyr Asn Thr Asn Pro Ser Gln Leu Lys Met Leu Lys Glu Ser Glu 885 890 895 tta agt tta atg agc agt gca agt agt tct gcc agc tct tcc aaa aaa 2736 Leu Ser Leu Met Ser Ser Ala Ser Ser Ser Ala Ser Ser Ser Lys Lys 900 905 910 tct tcg tct agg ata tcc acc ggg aca tta aag gac atg agt aat ctg 2784 Ser Ser Ser Arg Ile Ser Thr Gly Thr Leu Lys Asp Met Ser Asn Leu 915 920 925 tca aca tat gag gat ttg gcc cac cga acg aat aag ctt aag tat gaa 2832 Ser Thr Tyr Glu Asp Leu Ala His Arg Thr Asn Lys Leu Lys Tyr Glu 930 935 940 atc gga gat gat tct aga gca cat tct caa tct act ttg tcc gag cag 2880 Ile Gly Asp Asp Ser Arg Ala His Ser Gln Ser Thr Leu Ser Glu Gln 945 950 955 960 gaa caa gtt ccc ctg gaa aac gat aag gac agt ggc gag atg atg ctt 2928 Glu Gln Val Pro Leu Glu Asn Asp Lys Asp Ser Gly Glu Met Met Leu 965 970 975 gca gac ccc gaa atg aag cac aag tta gaa ttg gcc aga att tac tca 2976 Ala Asp Pro Glu Met Lys His Lys Leu Glu Leu Ala Arg Ile Tyr Ser 980 985 990 aga gat aaa tct caa ttt gtg aaa gaa gga aat ttt aaa gtt gac gaa 3024 Arg Asp Lys Ser Gln Phe Val Lys Glu Gly Asn Phe Lys Val Asp Glu 995 1000 1005 aat ttg att att agc aaa att tca ctt tcc cca agc act gaa tcc 3069 Asn Leu Ile Ile Ser Lys Ile Ser Leu Ser Pro Ser Thr Glu Ser 1010 1015 1020 tta gca gat tct aaa agt tct ggg aaa ggg ctt tct cca ctt gaa 3114 Leu Ala Asp Ser Lys Ser Ser Gly Lys Gly Leu Ser Pro Leu Glu 1025 1030 1035 gag gaa aag cta att gac gaa aac gct aca gaa aac gga tta gcg 3159 Glu Glu Lys Leu Ile Asp Glu Asn Ala Thr Glu Asn Gly Leu Ala 1040 1045 1050 gga tca cct aaa gac gaa gac gga atc ata atg act aac aag cga 3204 Gly Ser Pro Lys Asp Glu Asp Gly Ile Ile Met Thr Asn Lys Arg 1055 1060 1065 gga aac caa cct gtt agt act ttc cta cgc acc ccc gaa aag aac 3249 Gly Asn Gln Pro Val Ser Thr Phe Leu Arg Thr Pro Glu Lys Asn 1070 1075 1080 atc ggt gct caa aag cat gtt aag aag ttt tcg gac ttc gta agt 3294 Ile Gly Ala Gln Lys His Val Lys Lys Phe Ser Asp Phe Val Ser 1085 1090 1095 ctg caa aaa atg ggt gaa ggt gca tat ggt aag gtc aac cta tgt 3339 Leu Gln Lys Met Gly Glu Gly Ala Tyr Gly Lys Val Asn Leu Cys 1100 1105 1110 att cat aag aag aat agg tat att gtg gtg att aag atg att ttt 3384 Ile His Lys Lys Asn Arg Tyr Ile Val Val Ile Lys Met Ile Phe 1115 1120 1125 aaa gaa aga atc ctt gta gat aca tgg gtt aga gat agg aaa tta 3429 Lys Glu Arg Ile Leu Val Asp Thr Trp Val Arg Asp Arg Lys Leu 1130 1135 1140 ggc act ata cct tct gag atc caa att atg gcc acg ttg aac aaa 3474 Gly Thr Ile Pro Ser Glu Ile Gln Ile Met Ala Thr Leu Asn Lys 1145 1150 1155 aaa cca cat gag aat att tta cgg tta ctg gat ttt ttt gaa gac 3519 Lys Pro His Glu Asn Ile Leu Arg Leu Leu Asp Phe Phe Glu Asp 1160 1165 1170 gac gat tac tat tat atc gaa act ccc gta cat ggt gaa aca gga 3564 Asp Asp Tyr Tyr Tyr Ile Glu Thr Pro Val His Gly Glu Thr Gly 1175 1180 1185 tgt ata gat ctt ttc gat cta att gaa ttt aaa acc aac atg acc 3609 Cys Ile Asp Leu Phe Asp Leu Ile Glu Phe Lys Thr Asn Met Thr 1190 1195 1200 gaa ttt gaa gca aaa ttg ata ttc aag cag gtt gta gcg gga ata 3654 Glu Phe Glu Ala Lys Leu Ile Phe Lys Gln Val Val Ala Gly Ile 1205 1210 1215 aaa cat cta cac gac cag ggt att gtt cac aga gat atc aag gat 3699 Lys His Leu His Asp Gln Gly Ile Val His Arg Asp Ile Lys Asp 1220 1225 1230 gag aat gtt atc gta gat tct aaa ggc ttt gtt aag att att gat 3744 Glu Asn Val Ile Val Asp Ser Lys Gly Phe Val Lys Ile Ile Asp 1235 1240 1245 ttt gga tct gct gcg tat gtc aaa agc gga cca ttt gat gtt ttt 3789 Phe Gly Ser Ala Ala Tyr Val Lys Ser Gly Pro Phe Asp Val Phe 1250 1255 1260 gtt ggg aca ata gat tat gct gcc cct gaa gtc tta gga gga aac 3834 Val Gly Thr Ile Asp Tyr Ala Ala Pro Glu Val Leu Gly Gly Asn 1265 1270 1275 cct tat gag ggc caa cca cag gat att tgg gct att ggt att cta 3879 Pro Tyr Glu Gly Gln Pro Gln Asp Ile Trp Ala Ile Gly Ile Leu 1280 1285 1290 ttg tat acg gtg gtc ttc aaa gaa aac ccc ttc tac aat ata gat 3924 Leu Tyr Thr Val Val Phe Lys Glu Asn Pro Phe Tyr Asn Ile Asp 1295 1300 1305 gaa ata tta gaa ggc gac ctg aaa ttc aat aat gca gag gaa gtt 3969 Glu Ile Leu Glu Gly Asp Leu Lys Phe Asn Asn Ala Glu Glu Val 1310 1315 1320 agt gaa gat tgc att gag tta atc aag agt att ttg aac cgt tgc 4014 Ser Glu Asp Cys Ile Glu Leu Ile Lys Ser Ile Leu Asn Arg Cys 1325 1330 1335 gta ccg aag aga ccc acc att gac gac ata aat aac gac aaa tgg 4059 Val Pro Lys Arg Pro Thr Ile Asp Asp Ile Asn Asn Asp Lys Trp 1340 1345 1350 ttg gtt att tga 4071 Leu Val Ile 1355 6 1356 PRT yeast 6 Met Pro Tyr Ile Gly Ala Ser Asn Leu Ser Glu His Ser Phe Val Asn 1 5 10 15 Leu Lys Glu Lys His Ala Ile Thr His Lys Gly Thr Ser Ser Ser Val 20 25 30 Ala Ser Leu Gln Thr Pro Pro Ser Pro Asp Gln Glu Asn His Ile Asp 35 40 45 Asn Glu Leu Glu Asn Tyr Asp Thr Ser Leu Ser Asp Val Ser Thr Pro 50 55 60 Asn Lys Lys Glu Gly Asp Glu Phe Gln Gln Ser Leu Arg Asp Thr Phe 65 70 75 80 Ala Ser Phe Arg Lys Thr Lys Pro Pro Pro Pro Leu Asp Phe Glu Gln 85 90 95 Pro Arg Leu Pro Ser Thr Ala Ser Ser Ser Val Asp Ser Thr Val Ser 100 105 110 Ser Pro Leu Thr Asp Glu Asp Ile Lys Glu Leu Glu Phe Leu Pro Asn 115 120 125 Glu Ser Thr His Ser Tyr Ser Tyr Asn Pro Leu Ser Pro Asn Ser Leu 130 135 140 Ala Val Arg Leu Arg Ile Leu Lys Arg Ser Leu Glu Ile Ile Ile Gln 145 150 155 160 Asn Pro Ser Met Leu Leu Glu Pro Thr Pro Asp Asp Leu Pro Pro Leu 165 170 175 Lys Glu Phe Ala Gly Arg Arg Ser Ser Leu Pro Arg Thr Ser Ala Ser 180 185 190 Ala Asn His Leu Met Asn Arg Asn Lys Ser Gln Ile Trp Asn Thr Thr 195 200 205 Ser Ala Thr Leu Asn Ala Phe Val Asn Asn Thr Ser Ser Ser Ser Ala 210 215 220 Ala Ser Ser Ala Leu Ser Asn Lys Lys Pro Gly Thr Pro Val Phe Pro 225 230 235 240 Asn Leu Asp Pro Thr His Ser Gln Thr Phe His Arg Ala Asn Ser Leu 245 250 255 Ala Tyr Leu Pro Ser Ile Leu Pro Glu Gln Asp Pro Leu Leu Lys His 260 265 270 Asn Asn Ser Leu Phe Arg Gly Asp Tyr Gly Asn Asn Ile Ser Pro Glu 275 280 285 Arg Pro Ser Phe Arg Gln Pro Phe Lys Asp Gln Thr Ser Asn Leu Arg 290 295 300 Asn Ser Ser Leu Leu Asn Glu Arg Ala Tyr Gln Glu Asp Glu Thr Phe 305 310 315 320 Leu Pro His His Gly Pro Ser Met Asp Leu Leu Asn Glu Gln Arg Ala 325 330 335 Asn Leu Lys Ser Leu Leu Asn Leu Leu Asn Glu Thr Leu Glu Lys Asn 340 345 350 Thr Ser Glu Arg Ala Ser Asp Leu His Met Ile Ser Leu Phe Asn Leu 355 360 365 Asn Lys Leu Met Leu Gly Asp Pro Lys Lys Asn Asn Ser Glu Arg Asp 370 375 380 Lys Arg Thr Glu Lys Leu Lys Lys Ile Leu Leu Asp Ser Leu Ala Glu 385 390 395 400 Pro Phe Phe Glu His Tyr Asn Phe Ile Gly Asp Asn Pro Ile Ala Asp 405 410 415 Thr Asp Glu Leu Lys Glu Glu Ile Asp Glu Phe Thr Gly Ser Gly Asp 420 425 430 Thr Thr Ala Ile Thr Asp Ile Arg Pro Gln Gln Asp Tyr Gly Arg Ile 435 440 445 Leu Arg Thr Phe Thr Ser Thr Lys Asn Ser Ala Pro Gln Ala Ile Phe 450 455 460 Thr Cys Ser Gln Glu Asp Pro Trp Gln Phe Arg Ala Ala Asn Asp Leu 465 470 475 480 Ala Cys Leu Val Phe Gly Ile Ser Gln Asn Ala Ile Arg Ala Leu Thr 485 490 495 Leu Met Asp Leu Ile His Thr Asp Ser Arg Asn Phe Val Leu His Lys 500 505 510 Leu Leu Ser Thr Glu Gly Gln Glu Met Val Phe Thr Gly Glu Ile Ile 515 520 525 Gly Ile Val Gln Pro Glu Thr Leu Ser Ser Ser Lys Val Val Trp Ala 530 535 540 Ser Phe Trp Ala Lys Arg Lys Asn Gly Leu Leu Val Cys Val Phe Glu 545 550 555 560 Lys Val Pro Cys Asp Tyr Val Asp Val Leu Leu Asn Leu Asp Asp Phe 565 570 575 Gly Ala Glu Asn Ile Val Asp Lys Cys Glu Leu Leu Ser Asp Gly Pro 580 585 590 Thr Leu Ser Ser Ser Ser Thr Leu Ser Leu Pro Lys Met Ala Ser Ser 595 600 605 Pro Thr Gly Ser Lys Leu Glu Tyr Ser Leu Glu Arg Lys Ile Leu Glu 610 615 620 Lys Ser Tyr Thr Lys Pro Thr Ser Thr Glu Asn Arg Asn Gly Asp Glu 625 630 635 640 Asn Gln Leu Asp Gly Asp Ser His Ser Glu Pro Ser Leu Ser Ser Ser 645 650 655 Pro Val Arg Ser Lys Lys Ser Val Lys Phe Ala Asn Asp Ile Lys Asp 660 665 670 Val Lys Ser Ile Ser Gln Ser Leu Ala Lys Leu Met Asp Asp Val Arg 675 680 685 Asn Gly Val Val Phe Asp Pro Asp Asp Asp Leu Leu Pro Met Pro Ile 690 695 700 Lys Val Cys Asn His Ile Asn Glu Thr Arg Tyr Phe Thr Leu Asn His 705 710 715 720 Leu Ser Tyr Asn Ile Pro Cys Ala Val Ser Ser Thr Val Leu Glu Asp 725 730 735 Glu Leu Lys Leu Lys Ile His Ser Leu Pro Tyr Gln Ala Gly Leu Phe 740 745 750 Ile Val Asp Ser His Thr Leu Asp Ile Val Ser Ser Asn Lys Ser Ile 755 760 765 Leu Lys Asn Met Phe Gly Tyr His Phe Ala Glu Leu Val Gly Lys Ser 770 775 780 Ile Thr Glu Ile Ile Pro Ser Phe Pro Lys Phe Leu Gln Phe Ile Asn 785 790 795 800 Asp Lys Tyr Pro Ala Leu Asp Ile Thr Leu His Lys Asn Lys Gly Leu 805 810 815 Val Leu Thr Glu His Phe Phe Arg Lys Ile Gln Ala Glu Ile Met Gly 820 825 830 Asp Arg Lys Ser Phe Tyr Thr Ser Val Gly Ile Asp Gly Leu His Arg 835 840 845 Asp Gly Cys Glu Ile Lys Ile Asp Phe Gln Leu Arg Val Met Asn Ser 850 855 860 Lys Val Ile Leu Leu Trp Val Thr His Ser Arg Asp Val Val Phe Glu 865 870 875 880 Glu Tyr Asn Thr Asn Pro Ser Gln Leu Lys Met Leu Lys Glu Ser Glu 885 890 895 Leu Ser Leu Met Ser Ser Ala Ser Ser Ser Ala Ser Ser Ser Lys Lys 900 905 910 Ser Ser Ser Arg Ile Ser Thr Gly Thr Leu Lys Asp Met Ser Asn Leu 915 920 925 Ser Thr Tyr Glu Asp Leu Ala His Arg Thr Asn Lys Leu Lys Tyr Glu 930 935 940 Ile Gly Asp Asp Ser Arg Ala His Ser Gln Ser Thr Leu Ser Glu Gln 945 950 955 960 Glu Gln Val Pro Leu Glu Asn Asp Lys Asp Ser Gly Glu Met Met Leu 965 970 975 Ala Asp Pro Glu Met Lys His Lys Leu Glu Leu Ala Arg Ile Tyr Ser 980 985 990 Arg Asp Lys Ser Gln Phe Val Lys Glu Gly Asn Phe Lys Val Asp Glu 995 1000 1005 Asn Leu Ile Ile Ser Lys Ile Ser Leu Ser Pro Ser Thr Glu Ser 1010 1015 1020 Leu Ala Asp Ser Lys Ser Ser Gly Lys Gly Leu Ser Pro Leu Glu 1025 1030 1035 Glu Glu Lys Leu Ile Asp Glu Asn Ala Thr Glu Asn Gly Leu Ala 1040 1045 1050 Gly Ser Pro Lys Asp Glu Asp Gly Ile Ile Met Thr Asn Lys Arg 1055 1060 1065 Gly Asn Gln Pro Val Ser Thr Phe Leu Arg Thr Pro Glu Lys Asn 1070 1075 1080 Ile Gly Ala Gln Lys His Val Lys Lys Phe Ser Asp Phe Val Ser 1085 1090 1095 Leu Gln Lys Met Gly Glu Gly Ala Tyr Gly Lys Val Asn Leu Cys 1100 1105 1110 Ile His Lys Lys Asn Arg Tyr Ile Val Val Ile Lys Met Ile Phe 1115 1120 1125 Lys Glu Arg Ile Leu Val Asp Thr Trp Val Arg Asp Arg Lys Leu 1130 1135 1140 Gly Thr Ile Pro Ser Glu Ile Gln Ile Met Ala Thr Leu Asn Lys 1145 1150 1155 Lys Pro His Glu Asn Ile Leu Arg Leu Leu Asp Phe Phe Glu Asp 1160 1165 1170 Asp Asp Tyr Tyr Tyr Ile Glu Thr Pro Val His Gly Glu Thr Gly 1175 1180 1185 Cys Ile Asp Leu Phe Asp Leu Ile Glu Phe Lys Thr Asn Met Thr 1190 1195 1200 Glu Phe Glu Ala Lys Leu Ile Phe Lys Gln Val Val Ala Gly Ile 1205 1210 1215 Lys His Leu His Asp Gln Gly Ile Val His Arg Asp Ile Lys Asp 1220 1225 1230 Glu Asn Val Ile Val Asp Ser Lys Gly Phe Val Lys Ile Ile Asp 1235 1240 1245 Phe Gly Ser Ala Ala Tyr Val Lys Ser Gly Pro Phe Asp Val Phe 1250 1255 1260 Val Gly Thr Ile Asp Tyr Ala Ala Pro Glu Val Leu Gly Gly Asn 1265 1270 1275 Pro Tyr Glu Gly Gln Pro Gln Asp Ile Trp Ala Ile Gly Ile Leu 1280 1285 1290 Leu Tyr Thr Val Val Phe Lys Glu Asn Pro Phe Tyr Asn Ile Asp 1295 1300 1305 Glu Ile Leu Glu Gly Asp Leu Lys Phe Asn Asn Ala Glu Glu Val 1310 1315 1320 Ser Glu Asp Cys Ile Glu Leu Ile Lys Ser Ile Leu Asn Arg Cys 1325 1330 1335 Val Pro Lys Arg Pro Thr Ile Asp Asp Ile Asn Asn Asp Lys Trp 1340 1345 1350 Leu Val Ile 1355 7 3306 DNA yeast CDS (1)..(3303) 7 atg aca tac ccg gtt agt gca gcc gct cct gcc gac att tca tat tct 48 Met Thr Tyr Pro Val Ser Ala Ala Ala Pro Ala Asp Ile Ser Tyr Ser 1 5 10 15 aaa aat acc ccg ttg gta ggg ctc tct aaa cct cca tgc ttg tac caa 96 Lys Asn Thr Pro Leu Val Gly Leu Ser Lys Pro Pro Cys Leu Tyr Gln 20 25 30 cac gct tcc tcg tcc gtc gat tcg ttt tcg tca aca ttc agt gat gat 144 His Ala Ser Ser Ser Val Asp Ser Phe Ser Ser Thr Phe Ser Asp Asp 35 40 45 gat cgt tcg gac ctt gtt gct gta cct aat gag tcg ccg cat gca ttt 192 Asp Arg Ser Asp Leu Val Ala Val Pro Asn Glu Ser Pro His Ala Phe 50 55 60 tcg tat aat ccc ata tca cca aac tca ctg gga gta agg ttg acc atc 240 Ser Tyr Asn Pro Ile Ser Pro Asn Ser Leu Gly Val Arg Leu Thr Ile 65 70 75 80 tta aga agg tct ttg gaa ata atg gta aac agt cct gac atc tta cat 288 Leu Arg Arg Ser Leu Glu Ile Met Val Asn Ser Pro Asp Ile Leu His 85 90 95 gag ttg aag aaa aaa gca ccc gta ata gca tac ccc ccc tca ctt aga 336 Glu Leu Lys Lys Lys Ala Pro Val Ile Ala Tyr Pro Pro Ser Leu Arg 100 105 110 cac aca aga aac tta aca gag act gcc acg tta tca gca tcg cga gat 384 His Thr Arg Asn Leu Thr Glu Thr Ala Thr Leu Ser Ala Ser Arg Asp 115 120 125 ccg tta aat ggg tct cta att tca cca tta gta tcc aat atg cca tct 432 Pro Leu Asn Gly Ser Leu Ile Ser Pro Leu Val Ser Asn Met Pro Ser 130 135 140 cct gct agt aga ccc gtg ata cag aga gca acg tcc tta atg gtg ttg 480 Pro Ala Ser Arg Pro Val Ile Gln Arg Ala Thr Ser Leu Met Val Leu 145 150 155 160 cct gat aat gat act gcc agt aaa ctg aat cca gcc aag tcc gag tta 528 Pro Asp Asn Asp Thr Ala Ser Lys Leu Asn Pro Ala Lys Ser Glu Leu 165 170 175 gaa aac ttg tta ttc ttg cta aat tta gca ttg gaa aac aat tcc ttc 576 Glu Asn Leu Leu Phe Leu Leu Asn Leu Ala Leu Glu Asn Asn Ser Phe 180 185 190 gaa aga gct tcc gat tta cac atg cta tcg tta ttg aat atc aag aaa 624 Glu Arg Ala Ser Asp Leu His Met Leu Ser Leu Leu Asn Ile Lys Lys 195 200 205 ata aac ttt gat tca gac att caa aaa tca gaa act ttg aaa aag gtt 672 Ile Asn Phe Asp Ser Asp Ile Gln Lys Ser Glu Thr Leu Lys Lys Val 210 215 220 tta tta gat agt tta gca gaa cca ttt ttt gaa aac tac aag aag ttc 720 Leu Leu Asp Ser Leu Ala Glu Pro Phe Phe Glu Asn Tyr Lys Lys Phe 225 230 235 240 cct cac aaa gat tta ggt tcg aaa tcg caa tat aat gaa tat gag gag 768 Pro His Lys Asp Leu Gly Ser Lys Ser Gln Tyr Asn Glu Tyr Glu Glu 245 250 255 aaa cac gat gat ata gtc tcc tta gca gac atc aaa cca caa cag gac 816 Lys His Asp Asp Ile Val Ser Leu Ala Asp Ile Lys Pro Gln Gln Asp 260 265 270 tat agc cga att ctt cat cct ttc aca tct gca aaa aat tct ggt cca 864 Tyr Ser Arg Ile Leu His Pro Phe Thr Ser Ala Lys Asn Ser Gly Pro 275 280 285 gag gcc att ttt aca tgt tct caa caa tac ccc tgg aac ttc aaa gct 912 Glu Ala Ile Phe Thr Cys Ser Gln Gln Tyr Pro Trp Asn Phe Lys Ala 290 295 300 gcc aat gat ttg gcg tgt cta acg ttc ggt ata tca aag aat gtt atc 960 Ala Asn Asp Leu Ala Cys Leu Thr Phe Gly Ile Ser Lys Asn Val Ile 305 310 315 320 aag gca ctt act tta cta gac ctt att cat acg gat agc aga aat ttt 1008 Lys Ala Leu Thr Leu Leu Asp Leu Ile His Thr Asp Ser Arg Asn Phe 325 330 335 gtt cta gag aaa atc atg aat gca gaa gat gat aac caa gaa att gtg 1056 Val Leu Glu Lys Ile Met Asn Ala Glu Asp Asp Asn Gln Glu Ile Val 340 345 350 ttt acc gga gaa act ata cct atc gtc cag cca aat tcg aca agc aat 1104 Phe Thr Gly Glu Thr Ile Pro Ile Val Gln Pro Asn Ser Thr Ser Asn 355 360 365 aat aat gta cca aat ctg att tgg gct tca cta tgg gcc aag cgg aag 1152 Asn Asn Val Pro Asn Leu Ile Trp Ala Ser Leu Trp Ala Lys Arg Lys 370 375 380 aat ggc cta ctg gtc tgc gta ttt gaa aaa acg cct tgc gat tac att 1200 Asn Gly Leu Leu Val Cys Val Phe Glu Lys Thr Pro Cys Asp Tyr Ile 385 390 395 400 gac gtc atg tta aac ttg aga gat ttt tca gtg gac agc att att gat 1248 Asp Val Met Leu Asn Leu Arg Asp Phe Ser Val Asp Ser Ile Ile Asp 405 410 415 aca aca cac ttt ctg gaa aac ttt gac aag aaa aag cag caa gaa tca 1296 Thr Thr His Phe Leu Glu Asn Phe Asp Lys Lys Lys Gln Gln Glu Ser 420 425 430 act tcg cca atg aca gaa aag aaa acg gta aaa ttt gca aat gaa att 1344 Thr Ser Pro Met Thr Glu Lys Lys Thr Val Lys Phe Ala Asn Glu Ile 435 440 445 cac gat att ggg tca gta agt cac tca ctg agt aaa cta att gat gat 1392 His Asp Ile Gly Ser Val Ser His Ser Leu Ser Lys Leu Ile Asp Asp 450 455 460 gta cgt ttt gga aaa gtg ttt tct gca gat gat gat tta tta ccc ttg 1440 Val Arg Phe Gly Lys Val Phe Ser Ala Asp Asp Asp Leu Leu Pro Leu 465 470 475 480 tct atc agg gtg gca aat cat gtc aac gaa gag aga tat ttt acg ttg 1488 Ser Ile Arg Val Ala Asn His Val Asn Glu Glu Arg Tyr Phe Thr Leu 485 490 495 aat tgt cta tct gaa aat ata cca tgt gct gtt aca act tcc gta ttg 1536 Asn Cys Leu Ser Glu Asn Ile Pro Cys Ala Val Thr Thr Ser Val Leu 500 505 510 gaa aac gaa ata aaa tta aaa att cat agt ttg ccc tat cag gct ggg 1584 Glu Asn Glu Ile Lys Leu Lys Ile His Ser Leu Pro Tyr Gln Ala Gly 515 520 525 cta ttt att gtt gac agt cac act tta agt ctt tta agt ttc aac aaa 1632 Leu Phe Ile Val Asp Ser His Thr Leu Ser Leu Leu Ser Phe Asn Lys 530 535 540 tca gtc gcc aaa aac atg ttt ggc ttg cga ctt cac gag ttg gcc ggt 1680 Ser Val Ala Lys Asn Met Phe Gly Leu Arg Leu His Glu Leu Ala Gly 545 550 555 560 agt tcg gtt act aag ttg gtt cct tct ttg gcg gac atg ata tct tat 1728 Ser Ser Val Thr Lys Leu Val Pro Ser Leu Ala Asp Met Ile Ser Tyr 565 570 575 atc aat aaa act tat cct atg tta aat atc aca tta cca gaa aat aaa 1776 Ile Asn Lys Thr Tyr Pro Met Leu Asn Ile Thr Leu Pro Glu Asn Lys 580 585 590 gga ttg gtt tta aca gaa cat ttt ttc aga aaa att gag gct gaa atg 1824 Gly Leu Val Leu Thr Glu His Phe Phe Arg Lys Ile Glu Ala Glu Met 595 600 605 cat cat gat aag gac tcg ttt tac act tct att ggt cta gac ggc tgt 1872 His His Asp Lys Asp Ser Phe Tyr Thr Ser Ile Gly Leu Asp Gly Cys 610 615 620 cac aaa gat ggt aat ttg ata aag gtg gat gtt caa tta cgg gtc ttg 1920 His Lys Asp Gly Asn Leu Ile Lys Val Asp Val Gln Leu Arg Val Leu 625 630 635 640 aat acg aat gct gta tta tta tgg att aca cac tca aga gac gtg gtc 1968 Asn Thr Asn Ala Val Leu Leu Trp Ile Thr His Ser Arg Asp Val Val 645 650 655 att gaa aat tat acc acc gtt cct tcg cag cta ccg atg tta aag gag 2016 Ile Glu Asn Tyr Thr Thr Val Pro Ser Gln Leu Pro Met Leu Lys Glu 660 665 670 aac gaa att gat gtc gtt ggc agt aga ggt agt tcc agt gca tct tcc 2064 Asn Glu Ile Asp Val Val Gly Ser Arg Gly Ser Ser Ser Ala Ser Ser 675 680 685 aag aaa tct tcg gaa aaa att cct gtg aat act ttg aag gca atg gct 2112 Lys Lys Ser Ser Glu Lys Ile Pro Val Asn Thr Leu Lys Ala Met Ala 690 695 700 gat ctg tcg att agc tcc gct gaa acg att tct aat tca gac gat gaa 2160 Asp Leu Ser Ile Ser Ser Ala Glu Thr Ile Ser Asn Ser Asp Asp Glu 705 710 715 720 gta gac tta aat caa gtg aat gaa aaa cta cga gaa act tct tgc ggt 2208 Val Asp Leu Asn Gln Val Asn Glu Lys Leu Arg Glu Thr Ser Cys Gly 725 730 735 aaa gtg aga ggt atc gaa tct aat gac aac aat aac tat gac gat gac 2256 Lys Val Arg Gly Ile Glu Ser Asn Asp Asn Asn Asn Tyr Asp Asp Asp 740 745 750 atg aca atg gtt gat gat cct gag tta aaa cat aaa att gaa tta acg 2304 Met Thr Met Val Asp Asp Pro Glu Leu Lys His Lys Ile Glu Leu Thr 755 760 765 aaa atg tac acg cag gac aaa tca aaa ttt gta aag gac gac aac ttt 2352 Lys Met Tyr Thr Gln Asp Lys Ser Lys Phe Val Lys Asp Asp Asn Phe 770 775 780 aaa gtg gat gaa aaa ttt ata atg agg ata att gaa ccg ata aac gga 2400 Lys Val Asp Glu Lys Phe Ile Met Arg Ile Ile Glu Pro Ile Asn Gly 785 790 795 800 gaa gaa atc aaa aag gaa aca aat gag cta gac aaa aga aat tct act 2448 Glu Glu Ile Lys Lys Glu Thr Asn Glu Leu Asp Lys Arg Asn Ser Thr 805 810 815 tta aaa gct acg tac ttg act act cca gag gct aat ata ggc tca caa 2496 Leu Lys Ala Thr Tyr Leu Thr Thr Pro Glu Ala Asn Ile Gly Ser Gln 820 825 830 aag cgc ata aag aaa ttt tct gat ttt act att ctg caa gtg atg ggt 2544 Lys Arg Ile Lys Lys Phe Ser Asp Phe Thr Ile Leu Gln Val Met Gly 835 840 845 gag ggt gcg tac ggt aaa gtc aat tta tgc att cat aac aga gaa cac 2592 Glu Gly Ala Tyr Gly Lys Val Asn Leu Cys Ile His Asn Arg Glu His 850 855 860 tat atc gtg gtc atc aaa atg att ttc aaa gag agg att tta gta gac 2640 Tyr Ile Val Val Ile Lys Met Ile Phe Lys Glu Arg Ile Leu Val Asp 865 870 875 880 aca tgg gtg aga gat aga aaa tta ggt act ata cct tct gag atc caa 2688 Thr Trp Val Arg Asp Arg Lys Leu Gly Thr Ile Pro Ser Glu Ile Gln 885 890 895 att atg gcg acc ttg aat aaa aat tcc caa gaa aat atc ttg aag tta 2736 Ile Met Ala Thr Leu Asn Lys Asn Ser Gln Glu Asn Ile Leu Lys Leu 900 905 910 tta gat ttt ttt gaa gat gac gat tat tat tac att gaa aca ccg gtc 2784 Leu Asp Phe Phe Glu Asp Asp Asp Tyr Tyr Tyr Ile Glu Thr Pro Val 915 920 925 cat gga gaa act ggt agt att gat cta ttt gat gtt atc gaa ttc aaa 2832 His Gly Glu Thr Gly Ser Ile Asp Leu Phe Asp Val Ile Glu Phe Lys 930 935 940 aaa gat atg gtt gaa cat gaa gct aaa ctg gtg ttt aaa cag gta gtc 2880 Lys Asp Met Val Glu His Glu Ala Lys Leu Val Phe Lys Gln Val Val 945 950 955 960 gct agt ata aag cat tta cat gac caa gga att gtt cat aga gac ata 2928 Ala Ser Ile Lys His Leu His Asp Gln Gly Ile Val His Arg Asp Ile 965 970 975 aag gac gaa aat gtt att gtt gat tct cat ggc ttt gta aaa tta atc 2976 Lys Asp Glu Asn Val Ile Val Asp Ser His Gly Phe Val Lys Leu Ile 980 985 990 gat ttc ggt tcg gct gcc tat atc aag agt gga cca ttc gat gtt ttt 3024 Asp Phe Gly Ser Ala Ala Tyr Ile Lys Ser Gly Pro Phe Asp Val Phe 995 1000 1005 gtg gga aca atg gac tat gct gca cct gaa gtc ctt ggt ggt tcc 3069 Val Gly Thr Met Asp Tyr Ala Ala Pro Glu Val Leu Gly Gly Ser 1010 1015 1020 tct tac aaa ggt aaa cca caa gat att tgg gct ctt ggt gtg cta 3114 Ser Tyr Lys Gly Lys Pro Gln Asp Ile Trp Ala Leu Gly Val Leu 1025 1030 1035 ctt tat acc ata att tat aaa gag aat cct tat tac aac att gat 3159 Leu Tyr Thr Ile Ile Tyr Lys Glu Asn Pro Tyr Tyr Asn Ile Asp 1040 1045 1050 gaa atc ttg gaa ggt gaa tta agg ttc gat aaa tcg gaa cat gtc 3204 Glu Ile Leu Glu Gly Glu Leu Arg Phe Asp Lys Ser Glu His Val 1055 1060 1065 agt gaa gaa tgc atc agt ttg ata aaa aga att ttg aca aga gaa 3249 Ser Glu Glu Cys Ile Ser Leu Ile Lys Arg Ile Leu Thr Arg Glu 1070 1075 1080 gtt gat aaa aga cct act att gat gaa ata tat gaa gat aaa tgg 3294 Val Asp Lys Arg Pro Thr Ile Asp Glu Ile Tyr Glu Asp Lys Trp 1085 1090 1095 ctt aaa atc tag 3306 Leu Lys Ile 1100 8 1101 PRT yeast 8 Met Thr Tyr Pro Val Ser Ala Ala Ala Pro Ala Asp Ile Ser Tyr Ser 1 5 10 15 Lys Asn Thr Pro Leu Val Gly Leu Ser Lys Pro Pro Cys Leu Tyr Gln 20 25 30 His Ala Ser Ser Ser Val Asp Ser Phe Ser Ser Thr Phe Ser Asp Asp 35 40 45 Asp Arg Ser Asp Leu Val Ala Val Pro Asn Glu Ser Pro His Ala Phe 50 55 60 Ser Tyr Asn Pro Ile Ser Pro Asn Ser Leu Gly Val Arg Leu Thr Ile 65 70 75 80 Leu Arg Arg Ser Leu Glu Ile Met Val Asn Ser Pro Asp Ile Leu His 85 90 95 Glu Leu Lys Lys Lys Ala Pro Val Ile Ala Tyr Pro Pro Ser Leu Arg 100 105 110 His Thr Arg Asn Leu Thr Glu Thr Ala Thr Leu Ser Ala Ser Arg Asp 115 120 125 Pro Leu Asn Gly Ser Leu Ile Ser Pro Leu Val Ser Asn Met Pro Ser 130 135 140 Pro Ala Ser Arg Pro Val Ile Gln Arg Ala Thr Ser Leu Met Val Leu 145 150 155 160 Pro Asp Asn Asp Thr Ala Ser Lys Leu Asn Pro Ala Lys Ser Glu Leu 165 170 175 Glu Asn Leu Leu Phe Leu Leu Asn Leu Ala Leu Glu Asn Asn Ser Phe 180 185 190 Glu Arg Ala Ser Asp Leu His Met Leu Ser Leu Leu Asn Ile Lys Lys 195 200 205 Ile Asn Phe Asp Ser Asp Ile Gln Lys Ser Glu Thr Leu Lys Lys Val 210 215 220 Leu Leu Asp Ser Leu Ala Glu Pro Phe Phe Glu Asn Tyr Lys Lys Phe 225 230 235 240 Pro His Lys Asp Leu Gly Ser Lys Ser Gln Tyr Asn Glu Tyr Glu Glu 245 250 255 Lys His Asp Asp Ile Val Ser Leu Ala Asp Ile Lys Pro Gln Gln Asp 260 265 270 Tyr Ser Arg Ile Leu His Pro Phe Thr Ser Ala Lys Asn Ser Gly Pro 275 280 285 Glu Ala Ile Phe Thr Cys Ser Gln Gln Tyr Pro Trp Asn Phe Lys Ala 290 295 300 Ala Asn Asp Leu Ala Cys Leu Thr Phe Gly Ile Ser Lys Asn Val Ile 305 310 315 320 Lys Ala Leu Thr Leu Leu Asp Leu Ile His Thr Asp Ser Arg Asn Phe 325 330 335 Val Leu Glu Lys Ile Met Asn Ala Glu Asp Asp Asn Gln Glu Ile Val 340 345 350 Phe Thr Gly Glu Thr Ile Pro Ile Val Gln Pro Asn Ser Thr Ser Asn 355 360 365 Asn Asn Val Pro Asn Leu Ile Trp Ala Ser Leu Trp Ala Lys Arg Lys 370 375 380 Asn Gly Leu Leu Val Cys Val Phe Glu Lys Thr Pro Cys Asp Tyr Ile 385 390 395 400 Asp Val Met Leu Asn Leu Arg Asp Phe Ser Val Asp Ser Ile Ile Asp 405 410 415 Thr Thr His Phe Leu Glu Asn Phe Asp Lys Lys Lys Gln Gln Glu Ser 420 425 430 Thr Ser Pro Met Thr Glu Lys Lys Thr Val Lys Phe Ala Asn Glu Ile 435 440 445 His Asp Ile Gly Ser Val Ser His Ser Leu Ser Lys Leu Ile Asp Asp 450 455 460 Val Arg Phe Gly Lys Val Phe Ser Ala Asp Asp Asp Leu Leu Pro Leu 465 470 475 480 Ser Ile Arg Val Ala Asn His Val Asn Glu Glu Arg Tyr Phe Thr Leu 485 490 495 Asn Cys Leu Ser Glu Asn Ile Pro Cys Ala Val Thr Thr Ser Val Leu 500 505 510 Glu Asn Glu Ile Lys Leu Lys Ile His Ser Leu Pro Tyr Gln Ala Gly 515 520 525 Leu Phe Ile Val Asp Ser His Thr Leu Ser Leu Leu Ser Phe Asn Lys 530 535 540 Ser Val Ala Lys Asn Met Phe Gly Leu Arg Leu His Glu Leu Ala Gly 545 550 555 560 Ser Ser Val Thr Lys Leu Val Pro Ser Leu Ala Asp Met Ile Ser Tyr 565 570 575 Ile Asn Lys Thr Tyr Pro Met Leu Asn Ile Thr Leu Pro Glu Asn Lys 580 585 590 Gly Leu Val Leu Thr Glu His Phe Phe Arg Lys Ile Glu Ala Glu Met 595 600 605 His His Asp Lys Asp Ser Phe Tyr Thr Ser Ile Gly Leu Asp Gly Cys 610 615 620 His Lys Asp Gly Asn Leu Ile Lys Val Asp Val Gln Leu Arg Val Leu 625 630 635 640 Asn Thr Asn Ala Val Leu Leu Trp Ile Thr His Ser Arg Asp Val Val 645 650 655 Ile Glu Asn Tyr Thr Thr Val Pro Ser Gln Leu Pro Met Leu Lys Glu 660 665 670 Asn Glu Ile Asp Val Val Gly Ser Arg Gly Ser Ser Ser Ala Ser Ser 675 680 685 Lys Lys Ser Ser Glu Lys Ile Pro Val Asn Thr Leu Lys Ala Met Ala 690 695 700 Asp Leu Ser Ile Ser Ser Ala Glu Thr Ile Ser Asn Ser Asp Asp Glu 705 710 715 720 Val Asp Leu Asn Gln Val Asn Glu Lys Leu Arg Glu Thr Ser Cys Gly 725 730 735 Lys Val Arg Gly Ile Glu Ser Asn Asp Asn Asn Asn Tyr Asp Asp Asp 740 745 750 Met Thr Met Val Asp Asp Pro Glu Leu Lys His Lys Ile Glu Leu Thr 755 760 765 Lys Met Tyr Thr Gln Asp Lys Ser Lys Phe Val Lys Asp Asp Asn Phe 770 775 780 Lys Val Asp Glu Lys Phe Ile Met Arg Ile Ile Glu Pro Ile Asn Gly 785 790 795 800 Glu Glu Ile Lys Lys Glu Thr Asn Glu Leu Asp Lys Arg Asn Ser Thr 805 810 815 Leu Lys Ala Thr Tyr Leu Thr Thr Pro Glu Ala Asn Ile Gly Ser Gln 820 825 830 Lys Arg Ile Lys Lys Phe Ser Asp Phe Thr Ile Leu Gln Val Met Gly 835 840 845 Glu Gly Ala Tyr Gly Lys Val Asn Leu Cys Ile His Asn Arg Glu His 850 855 860 Tyr Ile Val Val Ile Lys Met Ile Phe Lys Glu Arg Ile Leu Val Asp 865 870 875 880 Thr Trp Val Arg Asp Arg Lys Leu Gly Thr Ile Pro Ser Glu Ile Gln 885 890 895 Ile Met Ala Thr Leu Asn Lys Asn Ser Gln Glu Asn Ile Leu Lys Leu 900 905 910 Leu Asp Phe Phe Glu Asp Asp Asp Tyr Tyr Tyr Ile Glu Thr Pro Val 915 920 925 His Gly Glu Thr Gly Ser Ile Asp Leu Phe Asp Val Ile Glu Phe Lys 930 935 940 Lys Asp Met Val Glu His Glu Ala Lys Leu Val Phe Lys Gln Val Val 945 950 955 960 Ala Ser Ile Lys His Leu His Asp Gln Gly Ile Val His Arg Asp Ile 965 970 975 Lys Asp Glu Asn Val Ile Val Asp Ser His Gly Phe Val Lys Leu Ile 980 985 990 Asp Phe Gly Ser Ala Ala Tyr Ile Lys Ser Gly Pro Phe Asp Val Phe 995 1000 1005 Val Gly Thr Met Asp Tyr Ala Ala Pro Glu Val Leu Gly Gly Ser 1010 1015 1020 Ser Tyr Lys Gly Lys Pro Gln Asp Ile Trp Ala Leu Gly Val Leu 1025 1030 1035 Leu Tyr Thr Ile Ile Tyr Lys Glu Asn Pro Tyr Tyr Asn Ile Asp 1040 1045 1050 Glu Ile Leu Glu Gly Glu Leu Arg Phe Asp Lys Ser Glu His Val 1055 1060 1065 Ser Glu Glu Cys Ile Ser Leu Ile Lys Arg Ile Leu Thr Arg Glu 1070 1075 1080 Val Asp Lys Arg Pro Thr Ile Asp Glu Ile Tyr Glu Asp Lys Trp 1085 1090 1095 Leu Lys Ile 1100 9 15 PRT human 9 Ala Met Ala Arg Ala Ala Ser Ala Ala Ala Leu Ala Arg Arg Arg 1 5 10 15 

What is claimed is:
 1. A composition comprising an isolated polypeptide, said polypeptide: (a) comprising SEQ ID NO:2; (b) comprising residues 1-89 of SEQ ID NO:2 and sufficient to elicit a human PASK (SEQ ID NO:2)-specific antibody in a heterologous host; (c) consisting of about residues 131-237 of SEQ ID NO:2; or (d) consisting of about residues 341-439 of SEQ ID NO:2.
 2. A composition according to claim 1, wherein the polypeptide comprises SEQ ID NO:2.
 3. A composition according to claim 1, wherein the polypeptide comprises residues 1-89 of SEQ ID NO:2 and is sufficient to elicit a human PASK (SEQ ID NO:2)-specific antibody in a heterologous host.
 4. A composition according to claim 1, wherein the polypeptide consists of about residues 131-237 of SEQ ID NO:2.
 5. A composition according to claim 1, wherein the polypeptide consists of about residues 341-439 of SEQ ID NO:2.
 6. A composition according to claim 1, wherein the polypeptide consists of residues 131-237 of SEQ ID NO:2.
 7. A composition according to claim 1, wherein the polypeptide consists of residues 341-439 of SEQ ID NO:2.
 8. A composition according to claim 1, further comprising ligand which binds the polypeptide.
 9. A method of detecting polypeptide-ligand binding comprising the steps of: (a) incubating a composition according to claim 8 under conditions wherein the polypeptide binds the ligand; and (b) detecting the binding of the polypeptide to the ligand.
 10. A method according to claim 9, wherein the composition further comprises a candidate modulator of the polypeptide-ligand binding.
 11. A method according to claim 9, wherein the polypeptide comprises SEQ ID NO:2, the ligand is a substrate, the composition further comprises a nucleoside triphosphate, the binding effects phosphorylation of the substrate, and the detecting step comprises detecting the phosphorylated substrate.
 12. A method according to claim 9, wherein the polypeptide consists of about residues 131-237 of SEQ ID NO:2 and the detecting step comprises detecting a change in NMR chemical shifts of the polypeptide and/or the ligand.
 13. A method according to claim 9, wherein the polypeptide consists of about residues 341-439 of SEQ ID NO:2 and the detecting step comprises detecting a change in NMR chemical shifts of the polypeptide and/or the ligand. 